Discovery of a 5 H-benzo[4,5]cyclohepta[1,2-b ]pyridin-5-one (MK-2461) inhibitor of c-Met kinase for the treatment of cancer

Article abstract of DOI:10.1021/jm200112k

c-Met is a transmembrane tyrosine kinase that mediates activation of several signaling pathways implicated in aggressive cancer phenotypes. In recent years, research into this area has highlighted c-Met as an attractive cancer drug target, triggering a number of approaches to disrupt aberrant c-Met signaling. Screening efforts identified a unique class of 5H-benzo[4,5] cyclohepta[1,2-b]pyridin-5-one kinase inhibitors, exemplified by 1. Subsequent SAR studies led to the development of 81 (MK-2461), a potent inhibitor of c-Met that was efficacious in preclinical animal models of tumor suppression. In addition, biochemical studies and X-ray analysis have revealed that this unique class of kinase inhibitors binds preferentially to the activated (phosphorylated) form of the kinase. This report details the development of 81 and provides a description of its unique biochemical properties.

Full text of DOI:10.1021/jm200112k

ARTICLE
pubs.acs.org/jmc
Discovery of a 5H-Benzo[4,5]cyclohepta[1,2-b]pyridin-5-one
MK-2461) Inhibitor of c-Met Kinase for the Treatment of Cancer
(
,†
†
†
†
†
Jason D. Katz,* James P. Jewell, David J. Guerin, Jongwon Lim, Christopher J. Dinsmore,
#
‡
‡
‡
||
§
Sujal V. Deshmukh, Bo-Sheng Pan, C. Gary Marshall, Wei Lu, Michael D. Altman, William K. Dahlberg,
‡
†
†
‡
‡
†
Lenora Davis, Danielle Falcone, Ana E. Gabarda, Gaozhen Hang, Harold Hatch, Rachael Holmes,
^
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‡
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#
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Kaiko Kunii, Kevin J. Lumb, Bart Lutterbach, Robert Mathvink, Naim Nazef, Sangita B. Patel,
^
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§
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†
Xianlu Qu, John F. Reilly, Keith W. Rickert, Craig Rosenstein, Stephen M. Soisson, Kerrie B. Spencer,
§
3
§
†
‡
Alexander A. Szewczak, Deborah Walker, Wenxian Wang, Jonathan Young, and Qinwen Zeng
†
‡
§
^
Departments of Chemistry, Oncology, In Vitro Pharmacology, Structural Chemistry, In Vivo Pharmacology,
#
3
Drug Metabolism and Pharmacokinetics, and Basic Pharmaceutical Sciences, Merck Research Laboratories,
3
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3 Avenue Louis Pasteur, BMB-2-114, Boston, Massachusetts 02115, United States
Global Structural Biology, Merck Research Laboratories, 770 Sumneytown Pike, West Point, Pennsylvania 19486, United States
S Supporting Information
b
ABSTRACT: c-Met is a transmembrane tyrosine kinase that mediates activation of
several signaling pathways implicated in aggressive cancer phenotypes. In recent years,
research into this area has highlighted c-Met as an attractive cancer drug target,
triggering a number of approaches to disrupt aberrant c-Met signaling. Screening
efforts identified a unique class of 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one kinase
inhibitors, exemplified by 1. Subsequent SAR studies led to the development of 81
(
MK-2461), a potent inhibitor of c-Met that was efficacious in preclinical animal models
of tumor suppression. In addition, biochemical studies and X-ray analysis have revealed
that this unique class of kinase inhibitors binds preferentially to the activated (phosphorylated) form of the kinase. This report
details the development of 81 and provides a description of its unique biochemical properties.
8
,9
’
INTRODUCTION
tyrosine kinase domain. Aberrant c-Met signaling, resulting from
MET genomic amplification, c-Met or HGF overexpression, or
c-Met mutations, is found in a variety of human cancers and often
c-Met, also referred to as hepatocyte growth factor receptor
(
HGFR), is a heterodimeric transmembrane receptor tyrosine
kinase composed of an extracellular R chain and a membrane
2,10
correlated with poor clinical outcomes. Recently, constitutive
c-Met activation due to MET amplificationwas foundto be a driver
of proliferation and survival of several gastric and lung cancer cell
lines and has been linked to acquired resistance of lung cancers
to epidermal growth factor receptor (EGFR) inhibitors.
In recent years, research has highlighted c-Met as an attractive
cancer drug target, triggering a number of approaches to disrupt
HGF/c-Met signaling. Both small-molecule c-Met kinase inhibi-
tors and antibodies targeting c-Met or HGF have exhibited
antitumor activities in preclinical models.
this report is to describe the discovery of a series of novel, ATP-
competitive, small molecule inhibitors of c-Met that preferen-
1,2
spanning β chain that are connected through a disulfide bond.
The tyrosine kinase domain resides in the cytosolic portion of the
11,12
1
,2
β chain. Binding of the endogenous ligand, hepatocyte growth
factor (HGF, also known as scatter factor), to c-Met induces
dimerization of the c-Met receptors and triggers transphosphor-
ylation (Y1234 and Y1235) and conformational changes that
13,14
2
initiate the kinase activity. The subsequent phosphorylation of a
short peptide sequence near the C-terminus (Y1349, Y1356, and
Y1365) forms a unique multisubstrate docking site that mediates
activation of several signaling pathways, including PI3K-PDK1-
15ꢀ30
The purpose of
2
ꢀ4
AKT-mTor, Ras-Rac-Pak, Ras-Raf-MEK-ERK, and PLC-γ. In
addition to the proliferative and antiapoptotic activities that are
common to many other growth factors, HGF/c-Met signaling
initiates unique motogenic and morphogenic phenotypes in c-Met
expressing cells by stimulating cellꢀcell detachment, migration,
31
tially bind to the activated form of c-Met.
Screening of the Merck compound collection identified N-(5-
oxo-3-phenyl-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl)meth-
anesulfonamide (compound 1, Figure 1) as a potent inhibitor of
c-Met, with an IC = 31 nM in our in vitro kinase assay. At the
time that this work was initiated, we were not aware of any
3
1
2,5,6
50
invasiveness, and formation and branching of tubules.
7
c-Met was first identified as the oncogenic fusion Tpr-Met. A
connection between c-Met and human cancer has been established
in hereditary papillary renal carcinoma (HPRC), which is causally
related to gain-of-function germline mutations in the c-Met
Received: January 31, 2011
Published: May 24, 2011
r 2011 American Chemical Society
4092
dx.doi.org/10.1021/jm200112k
|J. Med. Chem. 2011, 54, 4092–4108

Journal of Medicinal Chemistry
ARTICLE
Scheme 2. Synthesis of the 5H-Benzo[4,5]cyclohepta[1,2-b]-
a
pyridin-5-one Core Compounds 3aꢀe
Figure 1. Structure of sulfonamide 1.
reports indicating the use of 5H-benzo[4,5]cyclohepta[1,
2
-b]pyridin-5-one scaffolds as kinase inhibitors, which prompted
the team to investigate this lead in more detail. Additional in vitro
profiling indicated that 1 was an ATP competitive inhibitor of
c-Met (e.g., at 0.05 mM ATP, IC50 = 31 nM; at 0.10 mM ATP,
IC50 = 220 nM); however, initial molecular modeling experi-
ments did not provide a satisfactory hypothesis of its binding
mode in the ATP binding site. Further analysis indicated that
while 1 displayed moderate activity in a cell-biochemical assay
a
(
GTL-16 pY1349 IC = 2,500 nM), it showed no modulation of
(a) 6, KOt-Bu, THF, 0 ꢀC; (b) polyphosphoric acid, 200 ꢀC.
50
cell proliferation, a key phenotype of the c-Met pathway. This
was routinely measured in our screening funnel via measuring
proliferation of GTL-16 cells over a 72 h period (1, IC > 50000
a
Scheme 3. Incorporation of Amines into C Ring of 3
50
nM). Counterscreening against common off-target liabilities
revealed that 1 was not an inhibitor of three major CYP isozymes
(
3A4, 2C9, 2D6 IC50 > 50 μM), but there were some potential
concerns with regard to Kv11.1 inhibition (Kv11.1 IC
2
=
50
3
2
90 nM). Furthermore, 1 possessed suboptimal rat pharma-
cokinetic properties, with modest clearance (Cl = 24 mL/
p
min/kg), short half-life (t
= 30 min), and minimal oral
/2
1
33
bioavailability (F = 2%). In addition, the fused tricyclic core
of 1 contributed to its poor physical properties (log P = 3.15;
solubility at pH 7.4 = 0.5 μg/mL). The purpose of this report is to
detail our efforts to elucidate the structureꢀproperty relation-
ships about the central 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-
5
-one core of 1. In addition, details are provided regarding the
identification of compounds with increased potency and opti-
mized pharmacokinetic properties that demonstrate their poten-
tial as chemotherapeutic agents.
a
(
2 3
a) Benzophenone imine, Pd (dba) , NaOt-Bu, toluene, 110 ꢀC, 2.5 h;
then THF, 6N HCl; (b) 2,4-dimethoxybenzylamine, Pd (dba) , NaOt-
2
3
Bu, 1,4-dioxane, 100 ꢀC, 2.5 h; (c) TFA, DCM.
Scheme 1. Retrosynthetic Analysis of the 5H-Benzo-
formed via the condensation/dehydration of the substituted
-methyl nicotinates 5 with the appropriate bromobenzalde-
hydes 6. The following descriptions of the synthetic routes
[
4,5]cyclohepta[1,2-b]pyridin-5-one Core 2
2
(
Schemes 2ꢀ7) provide a concise overview of the methods
34
employed to access the target compounds.
As shown in Scheme 2, the core synthesis began with the base
induced condensation/dehydration of the substituted 2-methyl
35
nicotinates 5aꢀc, with the appropriate bromobenzaldehyde 6
to give the cyclization substrates 4aꢀd. Subsequent heating of
the functionalized 3-carboxyl-pyridines 4aꢀd in polyphosphoric
acid (PPA) at 200 ꢀC facilitated the intramolecular Friedelꢀ
Crafts cyclization to form the core seven-membered ring B in
3
6
3
aꢀe. In the case of the unsymmetrical bromide 4c, the
cyclization results in the formation of the regioisomers 3c and
’
CHEMISTRY
3d in approximately a 1:1 ratio.
A general retrosynthetic analysis for the construction of the
Upon formation of the tricyclic core, it was possible to
5
H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one core is presented
in Scheme 1. In this approach, the final substituted 5H-benzo-
4,5]cyclohepta[1,2-b]pyridin-5-ones 2 were prepared via palla-
derivatize the bromide on the C ring. As shown in Scheme 3,
the arylbromide 3 could be converted to the primary aniline via
palladium-mediated coupling with benzophenone imine, fol-
lowed by immediate acid-mediated hydrolysis of the intermedi-
[
dium catalyzed CꢀC and/or CꢀN bond forming events from
the bromo intermediates 3. The seven-membered core B of 3 was
available via the intramolecular FriedelꢀCrafts cyclization of the
37
ate imine to give 7 and 10. The bromide could also be
converted in a similar manner to a variety of amines, exemplified
by the conversion to the dimethoxybenzylamine derivative 8,
2
-vinylphenyl nicotinic acids 4. This key cyclization substrate was
4
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Journal of Medicinal Chemistry
ARTICLE
a
Scheme 4. Incorporation of Sulfonamides and Sulfamides into the C Ring
a
(
a) 3a or 3d, sulfonamide or sulfamide, Pd
2
(dba)
3
, Xantphos, Cs
2
CO
3
, 1,4-dioxane, 100 ꢀC; (b) 7 or 9, alkyl- or aryl-sulfonylchloride, NEt
3
, DCM; (c)
THF, 5N NaOH.
a
a
Scheme 5. Synthesis of Substituted Sulfamides
Scheme 7. Synthesis of N-Alkyl or N-Arylpyrazoles
a
9
(
a) R -X, base, CH CN or DMF; X = Cl, I, OMs, or OTf.
3
a
and did not couple effectively to elaborated sulfonyl or sulfamoyl
chlorides, which hindered an in-depth exploration about the
sulfonyl moiety (vide infra). Instead, a method was adopted that
involved direct conversion of the aryl bromide to the correspond-
ing arylsulfonamide (11, 12) or arylsulfamide (13) via a palla-
dium-mediated coupling with the corresponding primary
(
3
a) 15, NEt , DCM; (b) TFA, DCM.
a
Scheme 6. Functionalization of the A Ring
38
sulfonamide or sulfamide. The requisite sulfamides required
for the coupling were prepared from the corresponding second-
ary amines using the method described by Montero (14f16,
39
Scheme 5).
With the derivatization of the C ring complete, it was now
possible to functionalize the pyridyl ring (A ring). To that end,
the chloride in 12 or 13 could be converted to a substituted
aniline by utilizing various palladium-mediated amination stra-
40ꢀ42
tegies (Scheme 6, conditions a or b).
While both sets of
conditions delivered the desired aniline, condition a often resulted
in either significant proto-dehalogenation or reduction of the
carbonyl in the B ring. In contrast, for certain amine coupling
partners, condition b minimized these side reactions and would be
used instead. Similarly, the A ring pyridylchloride could be
converted to an aryl or heteroaryl derivative via palladium-
mediated cross-coupling with the corresponding boronic acid
or ester. While a number of conditions were explored for this
transformation (Scheme 6, conditions c, d, e, or f), those described
by Fu proved to be the most general and usually resulted in the
a
8
(
a) R -1ꢀ or 2ꢀ amine, Pd
2
(dba)
3
3
, NaOt-Bu, 1,4-dioxane, 100 ꢀC; (b)
8
8
R -1ꢀ or 2ꢀ amine, Pd (dba) , Cs CO , DMF, 180 ꢀC μw; (c) R -
boronic acid or ester, Pd(PPh
boronic acid or ester, PdCl
boronic acid or ester, Pd (dba)
2
2
3
8
3
)
4
, K
2
CO
3
, 1,4-dioxane, 100 ꢀC; (d) R -
3
8
2
(dppf), K
2
CO
3
, dioxane, 95 ꢀC; (e) R -
2
3
, P(t-Bu)
3
HBF
4
, KF, DMF, 100 ꢀC; (f)
8
R -boronic acid or ester, Pd (dba) , P(t-Bu) HBF , KF, DMF, 180 ꢀC
43
8
2
3
3
3
4
highest conversion to desired product.8 In a few cases (R = N-
substituted pyrazole), the required R boronic esters or acids
were not commercially available and one of two synthetic
strategies were adopted. In the first case, the chloride 13 was
converted to the boronic acid and subsequently coupled to the
corresponding halopyrazole. In the second strategy, the N-
substituted-pyrazole boronic esters or halides 20 were accessed
via the N-alkylation/arylation of the parent pyrazole 19 with a
μw; (g) (pinB)
2
, Pd
h) R -halide, Pd(PPh
Pd (dba) , P(t-Bu) HBF , KF, DMF, 100 ꢀC.
2
(dba)
3
, (C
6
H
11
)
3
P, KOAc, 1,4-dioxane, 100 ꢀC;
8
8
(
3 4 2 3
) , K CO , DMF, 185 ꢀC, μw; (i) R -halide
2
3
3
3
4
which could also be converted to the aniline 7 via treatment with
TFA (also 3bf9).
Similarly, the C ring bromide 3 could be converted into a wide
array of substituted sulfonamides and sulfamides via palladium-
mediated cross-coupling chemistry (Scheme 4). Initially, the
sulfonamides were installed by a stepwise approach, involving
bis-sulfonylation of the anilines (e.g., 7f12a), followed by
treatment with NaOH to affect partial hydrolysis of one sulfonyl
group to deliver the monoarylsulfonamides 12. At the outset of
the program, this approach allowed exploration of a wide array of
functionality on the C ring beyond the sulfonamide moiety found
in 1. However, anilines such as 7 proved to be poor nucleophiles
9
suitable R group (Scheme 7).
’
RESULTS AND DISCUSSION
Key Modifications of the Tricyclic Core of 1. Because the
5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one core of 1 had not
been described previously in the kinase literature, one of the
initial priorities was to explore the scaffold and its potential
4
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Journal of Medicinal Chemistry
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44
Table 1. SAR about the Central 5H-Benzo[4,5]cyclohepta-
c-Met are summarized in Table 1. Changes to the central seven-
membered ring resulted in a significant decrease in potency
against c-Met. Saturation of the double bond, as in 21, resulted in
a 70-fold drop in potency. Similarly, reduction of the carbonyl to
either the alcohol 22 or the methylene derivative 23 resulted in a
significant loss of activity. Further derivatization of 22 to tertiary
amines such as 24 delivered compounds more potent than the
parent alcohol but significantly less active than 1. At the outset of
this effort, the origins of this very narrow SAR were not well-
understood. However, molecular modeling results indicated that
the core of 1 had a high degree of planarity and all of these
modifications likely resulted in a central B ring that was sig-
nificantly puckered (vide infra). Given that 1 was an ATP
competitive inhibitor and likely bound to the hinge region of
the ATP binding site, it is reasonable to assume that significant
changes to the planarity of the core could impact ligand binding
affinity to c-Met.
[
1,2-b]pyridin-5-one Core of 1
The position of the aniline on the C ring was also important for
retaining c-Met activity. Moving the pharmacophore from the
7
-position to the 6- or 8-position, as with compounds 10 and 11,
resulted in c-Met IC50 values of 1100 nM and >10000 nM,
respectively (for a discussion of the aniline as a pharmacophore in
this scaffold, refer to Table 4). Simple alkylation of sulfonamide 1
(
N-methyl sulfonamide 25) led to a 25-fold loss in potency,
indicating that the sulfonamide NꢀH may be involved in key
hydrogen bonding interactions, although other possibilities
could be inferred (e.g., conformational bias, steric interaction
in the binding site, etc.). Along with other data not shown, these
results indicated that the original position of the sulfonamide
pharmacophore was optimal for c-Met activity.
Selected modifications of the A ring of 1 revealed a similarly
narrow SAR. Replacement or deletion of the phenyl ring, such as
in the aryl chloride 12a or the proto-derivative 27, resulted in a
loss of activity on the same order of magnitude observed for other
core alterations. However, the relatively moderate 12-fold loss in
potency for the chloride 12a indicated that this position might be
a promising site for additional modifications. In contrast, moving
the substitution to other positions on the pyridyl ring completely
eroded the activity such as with the 4-chloropyridine derivative
26. Finally, simple changes to the pyridine indicated that the N-1
atom was important for binding to c-Met, as conversion to the
2
1
-amino-pyridine derivative 28 or the N-oxide 29 led to a 200- and
00-fold loss in activity, respectively. The erosion of potency with
the N-oxide 29 would be expected if the pyridine was involved in
making key hinge interactions in the ATP binding pocket.
However, the results with 28 were more surprising, as amino-
pyridines making two hydrogen bonds to the hinge are common
in the kinase literature. Eventually, a crystal structure allowed
for a more complete analysis of this particular facet of the SAR
(
vide infra). Overall, the modifications detailed in Table 1
emphasized that the integrity of the tricyclic-heptenone core
of 1 needed to be maintained; however, they also revealed that
the phenyl and sulfonamide positions could be targeted for
further exploration.
Initial Substitutions at the 3-Position of the A Ring. An
initial goal of the early SAR about 1 was to identify compounds
that were more potent both in the primary HTRF c-Met assay as
well as the GTL-16 cell-biochemical assay. The GTL-16 cell line
is a gastric cancer cell line which harbors MET genomic
amplification and overexpresses constitutively activated c-Met
binding mode via targeted modifications around the periphery of
the 6,7,6-tricyclic core. The effects of some of these fundamental
structural changes to 1 on the ability to inhibit the function of
45
protein. For reasons that will be discussed below, inhibition in
this cell line was measured by monitoring levels of the C-terminal
4
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ARTICLE
Table 2. SAR of Amino Derivatives at the 3-Position
Table 3. SAR of Aryl and Heteroaryl Derivatives at the
-Position
3
31
docking site pY1349. The above modifications to the core of 1
Table 1) indicated that a reasonable initial strategy would be to
(
explore derivatives at the 3-position of the pyridine ring. Given
that the poor physical properties (e.g., solubility, log P, protein
binding) of 1 potentially contributed to its poor cellular activity,
amine analogues at the 3-pyridyl position were targeted to
address these issues (Table 2). Initial results appeared promising,
as the piperidinyl derivative 30 (ACD log P = 1.8) was 2.5-fold
more active in the cellular assay even though it displayed 3-fold
less intrinsic potency compared to that of 1 (ACD log P = 2.0). In
general, cyclic tertiary amines, such as 30 and the morpholino
analogue 31 displayed similar intrinsic c-Met activity compared
to 1 and exhibited a more favorable cell-shift. In contrast, tertiary
acyclic amines such as the N-butyl-N-methyl derivative 32
proved to be less active, although this was not general, as the
cyclopropylmethyl derivative 33 retained some of this potency.
One of the first compounds with promising activity in the cellular
assay was the isopropylpiperazine analogue 34 (ACD
log P = 1.5), which retained the intrinsic activity of 1 but was
nearly 20-fold more active in the cellular assay. Related amines,
such as the bicyclic ketal 35 or the 2-piperazinylpyridine 36,
showed notably increased cell shifts. While amino compounds
such as 34 satisfied the initial goal of identifying potent com-
pounds in the GTL-16 cell-biochemical assay, further character-
ization revealed that most of the amino analogues displayed poor
Aryl and heteroaryl derivatives at the 3-position of 1 were
being investigated in parallel with the amino analogues. As a
general trend, 2-substituted arenes were much less potent than
the 3- and 4-substituted analogues (e.g., 2-, 3-, and 4-chloro
analogues 37, 38, and 39, respectively), potentially by forcing the
phenyl group farther out of plane with the cycloheptenone core
(Table 3). Incorporation of six-membered heteroaryl groups,
such as the 4-pyridyl group in 40, improved both the solubility
and cell-shift, but related compounds such as the 3-pyridyl 41
and the pyrimidinyl 42 were not as well tolerated. On the other
hand, incorporation of five-membered heterocyclic analogues led
to a much more promising series of compounds. A significant
increase in both intrinsic c-Met activity and GTL-16 cell-shift was
observed in going from the 3-furanyl 43 to the 3-thienyl 44 and
finally the thiazolyl 45. Further screening of heterocycles, such as
the N-H pyrazole 46, revealed that this effect was not general;
however, we were pleased to observe that the N-methylpyrazolyl
47 displayed superior activity compared to any of the previous
compounds in the program and displayed only a 3-fold shift in
the cell-biochemical assay. In addition, 47 displayed improved
activity compared to historical compounds in the GTL-16 72 h
rat pharmacokinetics (typically Cl > Qhep). Ultimately, this
p
subseries was deprioritized in favor of new modifications
described below.
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Table 4. SAR at the Methanesulfonamide Position
a
Final compound was judged to be 85% pure; see Supporting Information for full details.
proliferation assay (IC50 = 1900 nM for 47 vs IC50 > 50000 nM
for 1). These encouraging results raised the possibility that 47
could be further evaluated in various in vivo models of c-Met
activity (e.g., tumor xenografts). Even though 47 exhibited
pyrazole was a key result (47 vs 46) that warranted further
investigation. Efforts to solve some of the issues with the scaffold via
various N-substituted pyrazoles are presented later in this report.
Initial SAR of the 7-Position. The initial SAR about the
core of 1 (Table 1) had indicated that the original 7-position of
the sulfonamide in 1 was preferred. As such, the team had begun
to explore analogues of the sulfonamide at the 7-position
concurrently with the above efforts on the 3-position. Initial
results revealed that the methane sulfonamide was not required
for c-Met activity. In fact, with an IC50 = 0.7 nM, the aniline
48 was approximately 7-fold more potent than the parent
sulfonamide 47 (Table 4). However, this change eroded the
cell-shift of the parent and 48 performed disappointingly in both
suboptimal rat pharmacokinetics (Cl = 40 mL/min/kg, t
=
p
1/2
3
0 min), it was the poor physical properties of the molecule
(solubility leading to formulation issues) that prevented the
execution of these experiments. Thus, while the N-methyl
pyrazole group greatly improved the overall activity of this class
of compounds, there were still many issues with the series yet to
be resolved. In addition to physical properties, some of the
detrimental off-target issues exhibited in 1, such as Kv11.1
activity, were still present in 47 (Kv11.1 IC = 10100 nM; IKr
5
0
32
patchclamp IC = 330 nM ). Regardless, the methylation of the
rat and dog pharmacokinetic studies, with Cl . Qhep. Close
5
0
p
4
097
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Journal of Medicinal Chemistry
ARTICLE
4
7
Table 5. SAR of N-substituted Pyrazoles
relative to the parent methane sulfonamide 47. Subsequent
3
sp hybridized carbon analogues revealed that there was room in
the binding site to extend out from the sulfonyl and still retain
potency against c-Met, such as with the 2-imidazoyl-ethyl
sulfonamide 58, but they lacked adequate potency in cellular
assays. On the other hand, incorporation of a nitrogen atom
adjacent to the sulfonyl, as with the sulfamide 59, resulted in a
compound that was equipotent to the methane sulfonamide 47.
Further characterization of 59 revealed that it had a nearly
identical compound profile to 47 (PK characteristics, cellular
activity, physical properties, etc.) with one key exception. Sulfa-
mide 59 did not have any of the Kv11.1 liabilities associated with
the methane sulfonamide (Kv11.1 IC > 30000 nM; IKr
5
0
46
patchclamp IC > 30000 nM). Because in vitro activity against
50
this ion channel had been a persistent problem with this series,
the sulfamide functionality offered an opportunity to both
explore new space in the binding region and attenuate off-target
liabilities and physical properties. This prospect was explored by
preparing two distinct sets of analogues of 59: (1) a series where
the sulfamide group was held constant and the pyrazole func-
tionality was varied, and (2) a series where the N-methyl pyrazole
was fixed and the sulfamide group was diversified.
Effect of N-substitution of the Pyrazole. Previous efforts
had identified N-methyl pyrazole as a more potent compound
than the corresponding N-H analogue (47 vs 46). To explore this
in more detail, an array of N-substituted pyrazoles were prepared to
see if further elaboration could attenuate the functional activity and
the pharmacokinetic properties of the parent sulfamide 59. Simple
alkyl substitution, such as the n-propyl analogue 60 (Table 5),
resulted in a slight boost in c-Met activity that also translated into
enhanced functional activity (GTL-16 proliferation). A range of
functional groups were well tolerated at this position, including
pendant amides (61), amines (62), and alcohols (63), potentially
due to this region extending out of the ATP binding pocket into a
solvent exposed region. In particular, the alcohol 63 was interesting
because it was one of the first compounds with an IC < 500 nM in
50
the GTL-16 proliferation assay; however, the pharmacokinetic
properties of all of these analogues were still suboptimal. Metabolite
ID of both 47 and 59 indicated that N-demethylation of the pyrazole
48
was one route for biotransformation of the parent. Consequently,
several strategies were investigated to address this issue, such as
preparing fluoroalkyl derivatives (64ꢀ67), constraining part of the
alkyl system into a ring (68), and direct attachment of a heteroaryl
substituent (69). Several of these analogues turned out to be some
of the most potent compounds prepared at the time. However,
these pyrazole analogues still suffered from poor pharmacokinetic
profiles and efforts were directed toward other strategies.
Structural Variation of the Sulfamide. While substitution of
the pyrazole did not identify a compound with a balanced profile
(potency, physical properties, pharmacokinetics), exploration of
the sulfamide of 59 proved to be more productive. Initial SAR
focused on small variations of the sulfamide framework (Table 6).
analogues of the aniline, such as the 2-chloro analogue 49,
N-Me aniline 50, and the phenol 51, were prepared for compar-
ison. Unfortunately, these compounds were significantly less
potent and exhibited a pharmacokinetic profile similar to the
aniline. Subsequent efforts uncovered a number of suitable
replacements for the sulfonamide and these other endeavors
will be reported in future communications. However, the rest of
this report will detail activities to develop the sulfonamide
pharmacophore.
Initial exploration of the methane sulfonamide itself was not at
all promising. While simple variations such as the ethylsulfon-
amide 52 were tolerated, other seemingly similar analogues such
as the vinyl sulfonamide 53 resulted in a loss of activity against
c-Met (Table 4). Indeed, it became apparent that sulfonamides
While both the ꢀNH analogue 70 and the monomethyl variant
2
71 retained the activity of the parent, other monosubstituted
sulfamides were less potent (data not shown). Conversely, N,N-
disusbstitution of the sulfamide was well-tolerated and a range of
activities was observed. Both the diethyl analogue 72 and the
pyrazinylethyl 73 retained good activity, in contrast to both the
benzyl 74 and the morpholinyl 75 analogues, which lost sig-
nificant potency against c-Met. Gratifyingly, most of the sulfa-
mide analogues retained their favorable Kv11.1 profile, although
these derivatives still displayed relatively poor rat pharmacokinetic
2
with a sp hybridized carbon adjacent to the sulfonyl were not
preferred, such as with the phenyl sulfonamide 54 or the
3
imidazoyl sulfonamide 55. On the other hand, sp hybridized
carbons adjacent to the sulfonyl were more consistently toler-
ated, as even the bulky tert-butyl sulfonamide 56 only suffered a
7
-fold loss in potency vs 47. The pK of the sulfonamide N-H also
a
seemed to be integral to the ability of the scaffold to bind to the
kinase, as the trifluoromethyl sulfonamide 57 was inactive
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Table 6. SAR of the N-Substituents of the Sulfamide
a
b
n = 3; dosed as a solution in DMSO:PEG400:water:ethanol (20:25:40:15); iv at 2 mg/kg; po at 4 mg/kg. n = 1; dosed as a solution in PEG400:40%
c
Captisol (35:65) with 1.2 equiv methanesulfonic acid; iv at 2 mg/kg. n = 2; dosed as a solution in propylene glycol:40% Captisol:water (25:55:20) with
1
acid; iv at 2 mg/kg. n = 2; dosed as a solution in propylene glycol:PEG400:40% Captisol (15:35:50); iv at 2 mg/kg. n = 3; dosed as a solution in DMSO:
PEG400:40% Captisol:water (10:20:50:20) with 2.2 equiv methanesulfonic acid; iv at 0.31 mg/kg and po at 0.62 mg/kg. n = 4; dosed as a solution in
d
.2 equiv KOH; iv and po at 1 mg/kg. n = 2; dosed as a solution in propylene glycol:PEG400:40% Captisol (15:40:45) with 1.2 equiv methanesulfonic
e
f
g
0.5% methylcellulose; iv at 2 mg/kg and po at 5 mg/kg.
properties. Further in vitro metabolite ID studies indicated that
monodealkylation of the sulfamide was a major route of
biotransformation, and it was believed that certain cyclic
analogues might reduce this metabolic pathway. Satisfyingly,
the azetidine analogues 76 and 77 displayed an improved rat
pharmacokinetic profile relative to 59, with low plasma clearance,
relative to 59. Unfortunately, these analogues were still limited by
micromolar IC50 values in cell-based functional assays. Even-
tually, it was found that cyclic ethers offered a balance between
acceptable cell-based activities and pharmacokinetic properties.
Screening a series of cyclic ethers led to the identification of the
4
9
31
(R)-dioxanyl analogue 81 (MK-2461 ), a potent inhibitor of the
c-Met. Compared to the initial tricyclic lead, 81 displayed superior
cell potency (GTL-16 IC = 56 nM, GTL-16 72 h proliferation =
acceptable V , and an improved half-life. In addition, certain
d
acyclic alkyl ethers (78) exhibited a similarly enhanced PK profile
50
4
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Journal of Medicinal Chemistry
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4
40 nM) and excellent rat pharmacokinetic properties (Cl =
signature was maintained in the GTL-16 cell line. Treatment
of these cells potently inhibited phosphorylation of Y1349 and
Y1365 in the c-Met C-terminal domain (IC50 ≈ 50 nM) and
inhibited phosphorylation of both AKT and ERK1/2, down-
p
10 mL/min/kg, t1/2 = 1.3 h). In general, 81 had a balanced PK
profile across species (Table 7), with moderate Cl and suitable
bioavailability, consistent with acceptable human PK predictions.
While high Cl was observed in the mouse, this did not hinder
profiling in animal models (vide infra). In addition to favorable PK
properties, 81 offered a suitable balance of off-target activities and
physical properties. It was free of Kv11.1 liabilities (Kv11.1 IC50
p
31
stream signaling molecules of c-Met pathway. In contrast,
treatment of these cells with 81 had almost no inhibitory effect
on phosphorylation of the c-Met activation loop residues Y1234
p
31
and Y1235 (IC50 > 2500 nM). The fact that 81 was inefficient
at inhibiting phosphorylation of the activation loop but was a
potent inhibitor of phosphorylation at other regions on the
protein suggested that it was preferentially binding to a form of
the kinase with an activated (phosphorylated) activation loop. A
second set of experiments designed to directly measure the
binding of 81 to both the phosphorylated and unphosphorylated
forms of the protein helped to confirm this hypothesis. Specifi-
cally, a series of BIAcore experiments revealed that these
compounds preferentially bind to the activated form of the
kinase (K = 4 nM) versus the unphosphorylated form (K =
>
30000 nM; I patchclamp IC > 30,000 nM), as well as minimal
Kr 50
potential for drugꢀdrug interactions (CYP3A4, 2C9, 2D6 IC
=
50
7
.1, 1.2, >100 μM, respectively). While optimizing from 1 to 81, the
log Pwas lowered from 3.15 to 2.70 and the ligand binding efficiency
LBE) only marginally decreased from 0.38 to 0.34. Finally, while
the solubility of the free base was only slightly improved to
(
3
1
μg/mL, various salt forms displayed solubility in excess of
00 mg/mL. Because of this balanced profile, as well as the
properties described below, 81 was ultimately selected as the first
clinical candidate for this program.
d
d
31
Unique Binding Mode of the Benzo[4,5]cyclohepta[1,2-
b]pyridin-5-one Scaffold. At the same time that the tricyclic
class was evolving toward a potent inhibitor of c-Met with
suitable drug-like properties, a series of in vitro biochemical
experiments identified some unique properties of this class of
compounds, as reported in a recent communication. While 81
was competitive with ATP and thus likely bound in the ATP
binding pocket, it displayed a unique signature of inhibition for
various phosphotyrosine residues on the kinase. In particular, in a
cell-free biochemical assay (cytosolic domain), 81 was more
effective at inhibiting phosphorylation of the C-terminal docking
25 nM). This data, combined with a series of molecular
modeling and cocrystal structures, allowed a complete picture
of the binding of these molecules and their mode of action to
emerge.
Crystal Structure of Tricyclic Inhibitor 82 Bound to the
Phosphorylated c-Met Kinase Domain. Recently, the crystal
structure of a structurally related tricyclic analogue of 81 bound to
the doubly autophosphorylated domain of c-Met, in which Y1234
31
50
and Y1235 are phosphorylated, was reported. This closely related
tricyclic analogue, identified as 83 in this report (Figure 2), displays
many of the binding properties ascribed to 81, such as preferential
binding to the autophosphorylated form of the kinase. However, it
contained a very different pharmacophore at the 7-position of
site residues Y1349 (IC50 ≈ 100 nM) and Y1365 (IC
≈
50
6 nM) than the autophosphorylation of the activation loop
2
50
residues Y1230/1234/1235 (IC50 ≈ 900 nM). This unique
the core (ethylene linked amide). In contrast, the tricyclic
sulfamide analogue 82 differs from 81 only in the pyrazole N-alkyl
substituent at the 3-position of the core (Figure 2).
Table 7. Pharmacokinetic Profile of 81
The X-ray crystal structure of 82 bound to the dually
phosphorylated kinase domain of c-Met was determined to
mouse
rat
dog
monkey
(
n = 2)
(n = 4)
(n = 4)
(n = 2)
2
.3 Å resolution (see Supporting Information Table S1). Upon
iv Parameters
binding 82, the kinase domain adopts a very similar overall
conformation to that observed upon binding 83. In both
a
b
b
a
50
dose (mg/kg)
1
2
1
0.25
AUC(0ꢀ¥) (μM h)
0.4
83
7.6
9.1
0.4
0.8
8.4
4.3
1.1
2.9
1.0
8.4
0.5
0.8
ligand-bound structures, Y1234 and Y1235 are phosphorylated,
the activation loop is ejected from the ATP binding site, and the
G loop adopts an extended induced-fit conformation that packs
against the ligand to form part of the binding site (Figure 3A).
The closely related analogues, 82 and 83, bind in the ATP
binding pocket in very similar orientations.
The structure of 82 bound to the autophosphorylated c-Met
kinase domain confirms many of the previous hypotheses of the
binding mode of this class of compounds (vide supra). In
particular, the pyridine nitrogen of the core forms a hydrogen
bond with the hinge backbone NH of M1160. In this orientation,
the pyrazole extends along the hinge and its piperdine substituent
projects out into solvent. Other key hydrogen bonds are made
3
Cl
p
(mL/min/kg)
Vdss (L/kg)
3.2
0.5
1
t
/2 (h)
po Parameters
a
c
c
a
dose (mg/kg)
2
5
2
0.5
AUC(0ꢀ¥) (μM h)
0.6
0.41
0.3
75
11.8
4.2
13.7
2.5
1.4
85
0.4
0.12
3
3
C
max (μM)
max (h)
bioavailability (%)
t
0.7
62
b
19
a
Dosed as a solution in 0.9% saline. Dosed as a solution in 0.5%
c
methylcellulose. Dosed in PEG400:captisol:water (25:50:25 (v/v/v)).
Figure 2. Ligands 82 and 83.
4
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Table 8. c-Met Mutant Profile of 81
enzyme
IC50 (nM)
WT c-Met
N1100Y
Y1230C
Y1230H
Y1235D
M1250T
2
1.5
1.5
1.0
0.5
0.4
Table 9. Kinase Selectivity Profile of 81
a
b
a
b
kinase
IC50 (nM)
kinase
IC50 (nM)
Figure 3. (A) Ribbon cartoon image of the X-ray crystal structure of 82
(
green) bound to activated c-Met kinase domain (gray). The activation
loop (magenta) and pY1234 and pY1235 (cyan) are also highlighted.
B) Binding pose of 82 in the ATP binding pocket.
Met
Ron
Flt1
Flt3
Mer
2
7
FGFR3
TrkB
FGFR1
Flt4
50
61
(
10
22
24
39
44
46
65
78
JAK2
Aur-A
Ret
230
290
640
7,800
between: (1) the aromatic NH of the sulfamide and the side
chain of D1222 and (2) one of the dioxanyl oxygens and the
backbone NH of R1086. As with the previously reported
structure of the complex with 83, the central carbonyl of the
core is involved in a water-mediated hydrogen bond to the more
proximal dioxanyl oxygen. Finally, the aromatic tricyclic core
adopts a conformation that is only 22ꢀ out of planarity, as
measured by the angle between the planes of the pyridyl and
phenyl rings about the central tropone ring.
FGFR2
KDR
TrkA
Abl
a
b
Human enzyme. Average of n = 2.
and (3) the relatively flat SAR observed in Table 5, as these
substituents are expected to be solvent exposed.
The binding mode for 82 agrees well with the observed general
SAR for the benzo[4,5]cyclohepta[1,2-b]pyridin-5-one chemical
series described in this report. For example, the slightly bent
geometry for the tricyclic core may be critical in allowing potent
analogues to bind in the ATP binding site (e.g., 1 and 81). In
contrast, compounds 21ꢀ24 are predicted by ab initio
Activity against Mutant Forms of c-Met. There have been
several reports in the literature of oncogenic mutations of
c-Met that confer resistance to small molecule inhibitors of
15,16,31,52,53
c-Met.
Because this tricyclic series of compounds
displayed a profound effect on c-Met signaling via residues outside
of the activation loop, while having little effect on activation loop
phosphorylation, the in vitro properties of 81 were compared
against some of these mutations. As can be seen in Table 8, 81
maintained potency against a spectrum of these mutations,
including several activation loop mutations. While there have been
no reports discussing these mutations with respect to treatment in
a clinical setting, it stands to reason that a compound that was
insensitive to acquired mutations in the kinase might be an
5
1
calculations to be locked into a more severely bent geometry,
likely due to reduction of the aromatic character of the central
7
-membered ring, thus precluding them from binding as effec-
tively in the ATP pocket. Additionally, the poor potency of
compounds 10, 11, and 25 can be explained by disruption of the
interaction between the sulfamide NH and the catalytic aspartate
side chain. Compounds 12a, 26, and 27 likely have reduced
activity due to the lack of hydrophobic packing interactions in the
hinge region that is provided by the phenyl or methylpyrazole
groups. Finally, compounds 28 and 29 are expected to disrupt the
hydrogen bond between the pyridine nitrogen and the backbone
of the hinge region. While it is obvious that the N-oxide in 29
would disrupt this bonding, it is less clear when considering 28.
In 82, the carbon at the 2-position is 2.9 Å from the carbonyl
oxygen of M1160, consistent with a potential CꢀH hydrogen
bonding interaction with the backbone carbonyl. As such,
5
2,54
advantage in the treatment of c-Met dependent tumors.
Kinase Selectivity Profile of 81. With any kinase inhibitor,
understanding the off-target kinase inhibition is crucial, especially
to help interpret both efficacy and potential side effects. In
general, the sulfonamide and sulfamide compounds in this report
display a similar kinase selectivity profile. As can be seen in
Table 9, 81 displays <100 nM activity against several other
kinases, including some that might prove beneficial in a clinical
setting, such as Ron, KDR, FGFR, and PDGFR-R. Even though
8
1 was found to be selective for the phosphorylated form of
substitution at the 2-position, such as with the ꢀNH of 28,
2
c-Met, this was shown to not be the case in several of the other
kinases in this panel. In particular, 81 was shown to inhibit
may result in a clash with the hinge. The observed binding
orientation also agrees well with many of the more subtle
modifications made throughout the lead optimization process,
including: (1) the preference for aryl groups at the 3-position
31
phosphorylation of the activation loop of both FGFR2 and
PDGFR, indicating that it is capable of binding to the unactivated
forms of these kinases. As such, while 81 is primarily an inhibitor
of c-Met, it is also a multikinase inhibitor. In particular, inhibition
of FGFR2 and PDGFR-R has potential implications for further
development of this series of compounds. When tested against a
panel of tumor cell lines, compound 81 was especially efficacious
at inhibiting cell lines that had either genomic amplification of
(
(
Tables 2 and 3) to maximize hydrophobic packing and planarity,
2) the tight SAR observed upon introduction of the sulfonam-
ide/sulfamide group (Table 4) likely due to the burial of this region
near the catalytic residues and the glycine rich loop (in compound
5
7, the sulfonamide nitrogen is likely deprotonated, leading to its
47
31
substantially poorer activity due to a loss of hydrogen bonding ),
c-Met or FGFR2 or constitutive activation of PDGFR.
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Figure 4. Compound 81 inhibited phosphorylation of c-Met and AKT and suppressed the growth of xenograft tumors in nude mice.
A) Inhibition by 81 of c-Met (Y1349) phosphorylation in GTL-16 tumors. The tumor bearing mice were given a single oral dose of 3, 10, 30, or
00 mg/kg of 81 and euthanized 1 h after dosing. The extent of c-Met (Y1349) phosphorylation in the tumors was determined by quantitative
(
1
Western blotting. Shown are representative images of phospho-c-Met (Y1349) and total c-Met blots. Also shown is the relationship between the
phospho-c-Met (Y1349) levels of all individual tumors and the corresponding host plasma total 81 concentrations. (B) The effects of orally
administered 81 on the growth of GTL-16 tumor. Asterisks indicate statistically significant difference from vehicle-treated group based on two-
way repeated measures ANOVA (* P e 0.05, ** P e 0.01, *** P e 0.001). (C) Immunostaining for phospho-c-Met (Y1349), phospho-c-Met
(
Y1365), and phospho-AKT (S473) in GTL-16 xenograft tumors following 21-day dosing with vehicle or 81. Tissues were collected at 4 h after
the last dose.
In Vivo Studies of 81. As discussed above, identifying
compounds in this series with an acceptable balance of potency,
physical properties, and pharmacokinetics was a challenge that
often hindered evaluation of the compounds in xenograft models.
However, several sulfamides with pendant ethers (e.g., 78, 79, 81)
displayed an improved pharmacokinetic profile that warranted
further evaluation of in vivo efficacy.
engagement (inhibition of pY1349) in vivo led to the evaluation
of 81 in a longer-term tumor xenograft efficacy study. In this
experiment, the compound was dosed orally (10, 50, and
100 mg/kg bid; 200 mg/kg qd) in a 21 day GTL-16 xenograft
study in CD1 nu/nu mice. As can be seen in Figure 4B, all doses
from this study resulted in statistically significant suppression of
tumor growth. The compound was well tolerated, and no change
in median body weight was observed (data not shown). As in the
acute PK/PD experiment, evidence of a dose-dependent inhibi-
tion of c-Met in the tumor was observed in this experiment. IHC
analysis of the tumor samples at 4 h postdose (Figure 4C)
showed inhibition of both c-Met and a downstream marker of
pathway inhibition, AKT. This indicates that even though 81
inhibits c-Met via a unique mode of action, it still displays an
inhibitory effect on the downstream pathway.
Cell-biochemical and cell-proliferation assays were conducted
in a GTL-16 cell line, a cell line dependent on c-Met for survival.
While the inhibition of GTL-16 cell proliferation and other ex vivo
readouts can indicate c-Met pathway inhibition, it was necessary to
establish c-Met dependent efficacy in vivo. In addition, it was not
known if the novel mode of action of this new class of kinase
inhibitors would translate to an in vivo model. A correlation
between in vitro/in vivo target engagement, pathway inhibition,
and efficacy was necessary to develop a strategy to eventually move
these compounds into the clinical setting. It should be noted that
while 81 had a relatively high plasma clearance and short half-life in
the mouse (see Table 7), it was still possible to achieve exposures
suitable for assessing efficacy in our mouse xenograft models.
As such, an acute PK/PD study of GTL-16 tumor-bearing
CD1 nu/nu mice was performed, with the animals receiving 81
orally at four doses (3, 10, 30, and 100 mg/kg). Inhibition of
c-Met was observed in vivo, with a plasma IC50 = 1.0 μM,
assessed at 1 h postdose (Figure 4A). This confirmation of target
’
CONCLUSION
The 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-ones have
been shown to be potent inhibitors of c-Met both in vitro and
in vivo. This series of compounds exemplify a unique type of
kinase inhibitors that preferentially bind to the phosphorylated
form of c-Met. Investigations into this series of compounds have
elucidated the structural basis of this selective inhibition and
identified the requirements for kinase inhibition. Incorporation of
4
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Journal of Medicinal Chemistry
ARTICLE
both the N-methyl pyrazole and the dioxanyl sulfamide function-
ality were crucial to achieving a balance between cell-based
activity and an acceptable pharmacokinetic profile. In addition,
the sulfamide functionality alleviated Kv11.1 activity inherent to
this structural class. Sulfamide 81 has a balanced pharmacokinetic
profile and has been shown to be efficacious in mouse tumor
xenograft experiments. Future communications will detail addi-
tional evaluation of this new series of kinase inhibitors.
1H), 7.44 (d, J = 12 Hz, 1H), 7.32 (d, J = 12.6 Hz, 1H). LRMS (ESI)
þ
calcd for (C14
H
BrClNO) [M þ H] 318.9; found 321.0.
8
General Procedure for the Conversion of 7-Bromo to
7-Amino Analogues. 7-Amino-3-chloro-5H-benzo[4,5]
cyclohepta[1,2-b]pyridin-5-one (7). Method A. A dry flask was
charged with 3a (3.0 g, 9.4 mmol), Pd (dba) (43 mg, 0.047 mmol), rac-
2
3
BINAP (88 mg, 0.14 mmol), NaOt-Bu (1.1 g, 11 mmol), 2,4-dimethox-
ylbenzylamine (1.4 mL, 9.4 mmol), and 1,4-dioxane (100 mL). The
mixture was sparged with argon for 10 min and then heated to 100 ꢀC for
2
h. The reaction mixture was cooled to ambient temperature and
concentrated in vacuo to afford a black oil. The black oil was slurried in
MeOH (20 mL) and DCM (80 mL). Heptane (300 mL) was added to the
mixture dropwise via addition funnel, and the mixture was stirred overnight.
’
EXPERIMENTAL SECTION
Chemistry. Commercial reagents were obtained from commercial
1
The resulting solid was collected by filtration to afford 2.6 g of 8 (68%). H
suppliers and used as received. All solvents were purchased in septum-
sealed bottles stored under an inert atmosphere. All reactions were
sealed with septa through which an argon atmosphere was introduced
unless otherwise noted. Liquid reagents and solvents were transferred
under a positive pressure of argon via syringe. Reactions were conducted
in microwave vials or round-bottomed flasks containing Teflon-coated
magnetic stir bars. Microwave reactions were performed with a Biotage
Initiator Series Microwave (fixed hold time setting; reaction tempera-
tures monitored by the internal infrared sensor).
NMR (600 MHz, DMSO) δ 8.91ꢀ8.89 (m, 1H), 8.48ꢀ8.46 (m, 1H), 7.54
(d, J = 8.4 Hz, 1H), 7.39 (s, 1H), 7.29 (d, J = 12.6 Hz, 1H), 7.11 (d, J = 7.8
Hz, 1H), 7.09ꢀ7.04 (m, 2H), 7.01 (d, J= 12.6 Hz, 1H), 6.58ꢀ6.56(m, 1H),
6
.46ꢀ6.43 (m, 1H), 4.25 (d, J = 5.4 Hz, 2H), 3.82 (s, 3H), 3.70 (s, 3H).
þ
LRMS (ESI) calcd for (C23
H
20ClN
2
O
3
) [M þ H] 407.1; found 407.0.
To a solution of 8 (50 mg, 0.12 mmol) in MeOH (1 mL) and DCM
(5 mL) was added TFA (1 mL). The reaction mixture was stirred at
4
0 ꢀC for 1 h. The reaction mixture was cooled to ambient temperature.
Reactions were monitored by thin layer chromatography (TLC) on
precoated TLC glass plates (silica gel 60 F254, 250 μm thickness) or by
LC/MS (30 mm ꢁ 2 mm 2 μm column þ guard; 2 μL injection; 3ꢀ98%
The mixture was diluted in EtOAc and washed with saturated aqueous
sodium bicarbonate. The organic layer was separated and dried over
MgSO . The solution was concentrated in vacuo and purified by flash
4
MeCN/H
2
O þ 0.05% TFA gradient over 2.3 min; 0.9 mL/min flow;
column chromatography (0ꢀ20% MeOH/DCM gradient) to afford
ESI; positive ion mode; UV detection at 254 nM). Visualization of the
developed TLC chromatogram was performed by fluorescence quench-
ing. Flash chromatography was performed on an automated purification
30 mg of 7 (95%).
Method B. A flask was charged with 3a (1.05 g, 3.30 mmol), Pd (dba)3
2
(8.0 mg, 0.008 mmol), racꢀrac-BINAP (15.0 mg, 0.025 mmol), NaOt-Bu
(0.444 g, 4.62 mmol), benzophenone imine (0.662 mL, 3.95 mmol), and
toluene (40 mL). The mixture was sparged with argon for 10 min then
heated to 110 ꢀC for 2.5 h. The reaction mixture was cooled to ambient
temperature and concentrated in vacuo. The solid was dissolved in 6N
HCl(aq) (1 mL) and THF (20 mL) and allowed to stir at ambient
temperature for 2 h. The mixture was diluted in EtOAc (300 mL) and
1
system using prepacked silica gel columns. H NMR were recorded on
either a 500 or 600 MHz Varian spectrometer; chemical shifts (δ) are
reported relative to residual protio solvent signals. Data for NMR spectra
are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet,
brs = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of
doublets, m = multiplet), coupling constant (Hz), integration.
All compounds reported are of at least 95% purity, as judged by LCAP
washed with saturated aqueous sodium bicarbonate (100 mL) and H O
2
(
150 mm ꢁ 4.6 mm ID, 5 μm column; 5 μL injection; 10ꢀ100%
(200 mL). The organic layer was separated and dried over MgSO . The
solution was concentrated in vacuo and purified by flash column chro-
4
MeCN/H
2
O þ 0.05% TFA gradient over 6.75 min; 1 mL/min flow;
ESI; positive ion mode; UV detection at 254 nM, Bw8), with the
exception of 56 (described in Supporting Information).
General Procedure for Formation of the Tricyclic Core.
7
5
matography (0ꢀ30% EtOAc/DCM gradient) to afford 700 mg of 7
1
(83%). H NMR (600 MHz, DMSO) δ 8.91 (d, J = 3.0 Hz, 1H), 8.48
(d, J = 3.0Hz, 1H), 7.52 (d, J = 10.8 Hz, 1H), 7.42 (d, J = 3.6Hz, 1H), 7.30
(d, J = 14.4 Hz, 1H), 7.03ꢀ6.99 (m, 2H), 6.17 (s, 2H). LRMS (ESI) calcd
-Bromo-3-chloro-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-
-one (3a). To a solution of methyl 5-chloro-2-methylnicotinate (3.50 g,
8.8 mmol) and p-bromobenzaldehyde (3.50 g, 18.8 mmol) in 500 mL of
þ
for (C H ClN O) [M þ H] 257.0; found 257.0.
14
10
2
1
General Procedure for the Incorporation of Sulfonamides.
N-(3-Chloro-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl)
methanesulfonamide (12a). Method A. To a solution of 7 (0.70 g,
2.7 mmol) and triethylamine (0.83 mL, 5.9 mmol) in EtOAc (40 mL) at
0 ꢀC was added methanesulfonyl chloride (0.42 mL, 5.4 mmol). The
reaction mixture was allowed to warm to ambient temperature and
stirred for 1 h. The mixture was diluted in EtOAc and washed with
hexanes at 0 ꢀC was added KOt-Bu (1.0 M in THF; 37.6 mL, 37.6 mmol).
The mixture was warmed to ambient temperature and THF (150 mL)
was added. The mixture was stirred at ambient temperature overnight.
The mixture was concentrated in vacuo to afford an orangeꢀyellow solid.
The solid was slurried in H O (50 mL) and 6 N aqueous HCl (30 mL).
2
EtOH (100 mL) was added to the mixture. The solid was collected by
filtration to afford 3.31 g of 4a (52%). H NMR (600 MHz, DMSO)
1
saturated aqueous sodium bicarbonate and H
2
O. The organic layer was
δ 13.79 (br s, 1H), 8.76 (d, J = 2.4 Hz, 1H), 8.22 (d, J = 3.0 Hz, 1H), 8.02
separated and dried over MgSO . The solution was concentrated in
4
(
d, J = 15.6 Hz, 1H), 7.79 (d, J = 15.6 Hz, 1H), 7.61ꢀ7.54 (m, 4H). LRMS
vacuo to afford a yellow solid. The yellow solid was dissolved in MeOH
(150 mL) and 5N NaOH(aq) (5 mL) and allowed to stir for 1 h. The
mixture was concentrated in vacuo to ∼10 mL. The mixture was diluted
81
þ
(ESI) calcd for (C14
H
10 BrClNO
2
) [M þ H] 340.0; found 339.9.
A flask was charged with 4a (35 g, 93 mmol) and 350 mL of
polyphosphoric acid (PPA). The mixture was heated to 200 ꢀC over-
night. The mixture was cooled to ambient temperature and then diluted
in ice. Solid NaOH was added to adjust to pH >10. The mixture was
diluted in DCM (8 L), Celite was added, and the mixture was stirred at
ambient temperature for 30 min. The solid was removed by filtration,
washing with DCM (4 L). The organic layer was separated and dried
in EtOAc (250 mL) and washed with saturated aqueous NH
4
Cl and
H O. The organic layer was separated and dried over MgSO . The
2 4
solution was concentrated in vacuo to afford 0.84 g of 12a (93%).
Method B. A flask was charged with 7 (5.00 g, 15.7 mmol),
methanesulfonamide (1.49 g, 15.7 mmol), Pd
2
(dba)
3
(0.714 g, 0.780
mmol), Xantphos (1.36 g, 2.35 mmol), Cs CO
(15.3 g, 47.0 mmol),
2
3
over MgSO
4
. The solution was concentrated in vacuo to afford 10.4 g of
and 1,4-dioxane (100 mL). The mixture was sparged with argon for
10 min and then heated to 95 ꢀC overnight. The reaction mixture was
cooled to ambient temperature and then diluted in EtOAc (2 L) and
1
3
a as a brown solid (35%). H NMR (600 MHz, DMSO) δ 8.98 (s, 1H),
8.42 (s, 1H), 8.19 (s, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 8.4 Hz,
4
103
dx.doi.org/10.1021/jm200112k |J. Med. Chem. 2011, 54, 4092–4108

Journal of Medicinal Chemistry
washed with H O (1 L). The organic layer was separated, washed with
ARTICLE
2
with argon for 10 min and then heated to 100 ꢀC overnight. The
reaction mixture was cooled to ambient temperature. The mixture was
brine (500 mL), and dried over MgSO . The solution was concentrated
4
in vacuo to afford a crude solid that was dissolved in 3:1 hot DCM/
MeOH (150 mL) and stirred for 2 h. Hexanes (150 mL) was added
dropwise via addition funnel, and the mixture was allowed to stir
overnight. Additional hexanes (50 mL) was added. After stirring for
diluted in EtOAc and washed with H
The organic layer was separated and dried over MgSO
was filtered, concentrated in vacuo, and purified by reverse phase
HPLC (40ꢀ100% CH CN/H O with 0.05% TFA) to afford 35 mg of
37 (71%). H NMR (600 MHz, DMSO-d ) δ 10.40 (s, 1H), 9.03 (d, J =
2
O and saturated aqueous NH
4
Cl.
4
. The solution
3
2
1
4
h, the solids were collected by filtration to afford 3.65 g of 12a (70%).
6
1
H NMR (600 MHz, DMSO) δ 10.42 (s, 1H), 8.97 (dd, J = 2.4 Hz,
1.8 Hz, 1H), 8.51 (d, J = 2.4 Hz, 1H), 8.02 (d, J = 2.4 Hz, 1H), 7.83 (d,
J = 8.4 Hz, 1H), 7.66ꢀ7.58 (m, 3H), 7.52ꢀ7.48 (m, 2H), 7.46 (d, J =
1.2 Hz, 1H), 8.48 (d, J = 2.4 Hz, 1H), 8.00 (d, J = 2.4 Hz, 1H), 7.80 (d, J =
7.8 Hz, 1H), 7.62 (dd, J = 8.4, 2.4 Hz, 1H), 7.42 (d, J = 12.0 Hz, 1H), 7.23
12.0 Hz, 1H), 7.31 (d, J = 12.0 Hz, 1H), 3.09 (s, 3H). LRMS (ESI)
þ
(
d, J= 12.0 Hz, 1H), 3.09 (s, 3H). LRMS (ESI) calcd for (C H ClN O S)
calcd for (C21
H
16ClN
2
O
3
S) [M þ H] 411.1; found 411.0.
15
12
2 3
þ
N-(3-Isothiazol-4-yl-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]
pyridin-7-yl)methanesulfonamide (45). Step 1. A flask was
charged with 12a (1.12 g, 3.34 mmol), bis(pinacolato)diboron (1.36 g,
[M þ H] 335.0; found 335.1.
N-(5-Oxo-3-phenyl-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-
-yl)methanesulfonamide (1). Compound 3b (200 mg,
7
2 3
5.35 mmol), Pd (dba) (0.160 g, 0.170 mmol), tricyclohexylphosphine
0
(
.552 mmol), methanesulfonamide (57.8 mg, 0.607 mmol), Pd
25.3 mg, 0.028 mmol), Xantphos (47.9 mg, 0.083 mmol), and
Cs CO (540 mg, 1.656 mmol) were combined in 1,4-dioxane
5.5 mL) and sparged with argon for 10 min. The reaction mixture
2 3
dba
(0.120 g, 0.400 mmol), potassium acetate (0.820 g, 8.35 mmol), and 1,4-
dioxane (50mL). Themixture wasspargedwith argon for 10minandthen
heated to 100 ꢀC overnight. The reaction mixture was cooled to ambient
temperature. The mixture wasdilutedin EtOAcandwashedwithsaturated
aqueous sodium bicarbonate. The organic layer was separated and dried
2
3
(
was heated to 100 ꢀC and stirred for 2 h then cooled to ambient
temperature. The reaction mixture was diluted in EtOAc, washed
with saturated aqueous sodium bicarbonate and brine, and then
dried over sodium sulfate, filtered, and concentrated. The residue
was purified by reverse phase HPLC (CH CN/H O with 0.1%
overMgSO
4
. Thesolution wasfiltered, concentratedinvacuo, andpurified
CN/H O with 0.1% TFA). The
by reverse phase HPLC (10ꢀ100% CH
3
2
fractions were diluted in EtOAc (500 mL) and washed with saturated
aqueous sodium bicarbonate (100 mL). The organic layer was separated,
3
2
TFA). The fractions containing product were combined and diluted
in EtOAc (200 mL) and aqueous sodium bicarbonate (100 mL) and
heated to 70 ꢀC so that all of the solids went into solution. The layers
were separated, the organic layer was washed with brine, and then
the combined aqueous layers were back-extracted with EtOAc. The
combined organic layers were dried over sodium sulfate. The
solution was filtered and concentrated under reduced pressure to
4
dried overMgSO , and filteredand concentrated in vacuo to afford 0.600 g
of {7-[(methylsulfonyl)amino]-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]-
1
pyridin-3-yl}boronic acid (52%). H NMR (600 MHz, CDCl ) δ 9.16
3
(
(
d, J = 1.8 Hz, 1H), 8.88 (d, J = 1.8 Hz, 1H), 7.91 (d, J = 2.4 Hz, 1H), 7.68
dd, J =7.8, 3.0 Hz, 1H), 7.61 (d, J=7.8 Hz, 1H), 7.35(d, J =12.0 Hz, 1H),
7
.28ꢀ7.21 (m, 2H), 3.08 (s, 3H). LRMS (ESI) calcd for (C15
14
H -
þ
1
BN O S) [M þ H] 345.1; found 345.1.
2 5
give 136 mg of compound 1 (65%) as a yellow solid. H NMR (600
Step 2. A reaction vessel was charged with {7-[(methylsulfonyl)
amino]-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-3-yl}boronic acid
MHz, DMSO-d
6
) δ 10.42 (br s, 1H), 9.24 (s, 1H), 8.64 (s, 1H), 8.03
(
s, 1H), 7.85 (s, 1H), 7.83 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.61 (d,
(
(
(
50 mg, 0.15 mmol), 4-bromo-isothiazole (48 mg, 0.29 mmol), Pd-
PPh ) (5.0 mg, 0.004 mmol), K CO (60 mg, 0.44 mmol), and DMF
2 mL). The mixture was sparged with argon for 10 min then placed in the
J = 8.4 Hz, 1H), 7.53ꢀ7.49 (m, 2H), 7.46ꢀ7.42 (m, 1H), 7.38 (d, J =
1
2.6 Hz, 1H), 7.26 (d, J = 12.0 Hz, 1H), 3.09 (s, 3H). LRMS (ESI)
3 4
2
3
þ
calcd for (C21
H
17
N
2
O
3
S) [M þ H] 377.1; found 377.1.
microwave reactor and heated to 185 ꢀC for 20 min. The reaction mixture
was cooled to ambient temperature. The mixture was diluted in EtOAc
General Procedure for the Incorporation of Amines: N-[3-
4-Isopropylpiperazin-1-yl)-5-oxo-5H-benzo[4,5]cyclohepta-
1,2-b]pyridin-7-yl]methanesulfonamide (34). A flask was
(
[
and washed with saturated aqueous NH Cl. The organic layer was
4
4
separated and dried over MgSO . The solution was filtered, concentrated
charged with 12a (50 mg, 0.15 mmol), 4-dimethylamino-piperdine
in vacuo, and purified by reverse phase HPLC (20ꢀ100% CH CN/H O
(
(
(
38 mg, 0.30 mmol), Pd (dba) (1.5 mg, 0.002 mmol), rac-BINAP
3.0 mg, 0.005 mmol), NaOt-Bu (43 mg, 0.45 mmol), and 1,4-dioxane
3 mL). The mixture was sparged with argon for 10 min and then heated
3
2
2
3
1
with 0.1% TFA) to afford 11 mg of 45 (20%). H NMR (600 MHz,
DMSO-d ) δ 10.62ꢀ10.18 (bs, 1H), 9.70 (s, 1H), 9.38 (d, J = 1.8 Hz,
6
1H), 9.26 (s, 1H), 8.78 (d, J = 2.4 Hz, 1H), 8.00 (d, J = 2.4 Hz, 1H), 7.78
to 105 ꢀC overnight. The reaction mixture was cooled to ambient
(d, J = 8.4 Hz, 1H), 7.59 (dd, J = 8.4, 2.4 Hz, 1H), 7.39 (d, J = 12.0 Hz,
temperature, diluted in EtOAc, and washed with H O and saturated
2
1
H), 7.26 (d, J = 12.6 Hz, 1H), 3.08 (s, 3H). LRMS (ESI) calcd for
aqueous NH
MgSO . The solution was concentrated in vacuo and purified by reverse
CN/H O with 0.1% TFA) to afford 15 mg
24%) of 34. H NMR (600 MHz, DMSO-d ) δ 10.28 (s, 1H), 8.76
4
Cl. The organic layer was separated and dried over
þ
(C
18
H
14
N
3
O
3
S
2
) [M þ H] 384.0; found 384.0.
4
0
N,N-Dimethyl-N -[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-
benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl]sulfuric Diamide
(59). Step 1. N -(3-Dhloro-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]-
phase HPLC (20ꢀ100% CH
3
2
1
(
(
(
6
0
d, J = 3.0 Hz, 1H), 8.01 (d, J = 2.4 Hz, 1H), 7.79 (d, J = 3.0 Hz, 1H), 7.73
d, J = 9.0 Hz, 1H), 7.57 (dd, J = 8.4, 2.4 Hz, 1H), 7.20ꢀ7.14 (m, 2H),
pyridin-7-yl)-N,N-dimethylsulfuric Diamide (13a). Compound 3a
(11.93 g, 37.21 mmol) and N,N-dimethylsulfuric diamide (4.62 g,
37.2 mmol) were combined in 1,4-dioxane (350 mL), and the solution
was sparged with argon for 1 h. Pd dba (1.70 g, 1.86 mmol), Xantphos
3
0
4
.38ꢀ3.34 (m, 4H), 3.05 (s, 3H), 2.72 (m, 1H), 2.62ꢀ2.56 (bs, 4H),
þ
.99 (d, J= 7.2 Hz, 6H). LRMS (ESI) calcd for (C H N O S) [M þ H]
2
2 27 4 3
27.2; found 427.2.
2
3
1
Compound 14 was isolated as a side-product from this reaction. H
2 3
(3.23 g, 5.58 mmol), and Cs CO (36.37 g, 111.6 mmol) were added, and
NMR (600 MHz, DMSO-d ) δ 8.92ꢀ8.90 (m, 1H), 8.45 (d, J = 7.8 Hz,
the reaction mixture was sparged with argon for an additional 30 min. The
reaction was then heated to 100 ꢀC for 2 h, during which time a thick
precipitate formed. After cooling to ambient temperature, the reaction
mixture was diluted in EtOAc (1.5 L) and washed with saturated aqueous
sodium bicarbonate (2 ꢁ 300 mL) and brine (300 mL). The combined
aqueouslayers were filteredthrough a padof Celite, andthe Celite pad was
washed with EtOAc (500 mL). The aqueous layer was combined with this
Celite EtOAc wash in a separatory funnel and then further washed with
300 mL of EtOAc. The combined organic layers were dried over sodium
6
1
H), 7.97 (d, J = 2.4 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.62ꢀ7.58 (m,
2
H), 7.39 (d, J = 12.6 Hz, 1H), 7.23 (d, J = 12.6 Hz, 1H), 3.06 (s, 3H).
þ
LRMS (ESI) calcd for (C15
H
13
N
2
O
3
S) [M þ H] 301.1; found 301.1.
N-[3-(2-Chlorophenyl)-5-oxo-5H-benzo[4,5]cyclohepta-
1,2-b]pyridin-7-yl]methanesulfonamide (37). A reaction ves-
[
sel was charged with 12a (40.0 mg, 0.12 mmol), 2-chlorophenylboro-
nic acid (22 mg, 0.14 mmol), Pd(PPh (4.0 mg, 0.004 mmol), K CO
50 mg, 0.36 mmol), and 1,4-dioxane (3 mL). The mixture was sparged
3
)
4
2
3
(
4
104
dx.doi.org/10.1021/jm200112k |J. Med. Chem. 2011, 54, 4092–4108

Journal of Medicinal Chemistry
ARTICLE
sulfate and then filtered and concentrated in vacuo to give a brown solid.
Thissolidwas dissolved in boiling10% MeOH/DCM(800 mL), andthen
hexanes were slowly added (500 mL) until a precipitate began to form
from the stirring solution. The mixture was allowed to cool to ambient
temperature, and then additional hexanes (3 ꢁ 250 mL aliquots at 30 min
intervals) were added and the mixture was left to stand for an additional
under an atmosphere of hydrogen (balloon) and stirred vigorously. After
3 h, the solution was filtered through Celite, concentrated in vacuo, and
azeotroped with toluene to afford the crude residue. The residue was
dissolved in 4.0 M HCl in 1,4-dioxane (20 mL) and concentrated in vacuo
to afford 5.37 g of 1-[(2R)-1,4-dioxan-2-yl]-N-methylmethanamine
1
hydrochloride (100%). H NMR (600 MHz, DMSO-d
): δ 8.94 (s,
6
1
h. The solids were collected by filtration and dried under vacuum to
1H), 8.75 (s, 1H), 3.86ꢀ3.80 (m, 1H), 3.76 (d, J = 11.6 Hz, 1H), 3.70 (d,
J = 11.5 Hz, 1H), 3.64 (d, J = 11.6 Hz, 1H), 3.58 (dt, J = 11.3, 2.3 Hz, 1H),
3.45 (dt, J = 11.2, 2.4 Hz, 1H), 3.22 (t, J = 10.6 Hz, 1H), 2.97ꢀ2.91 (m,
1H), 2.91ꢀ2.85 (m, 1H), 2.50 (d, J = 5.2, 3H).
afford 8.10 g of compound 13a (60%). If desired, additional 13a could be
recovered from the mother liquor via flash column chromatography. H
1
NMR (600 MHz, DMSO-d
6
) δ 10.55 (s, 1H), 8.96 (d, J = 2.4 Hz, 1H),
.49 (d, J = 2.9 Hz, 1H), 7.99 (d, J = 2.4 Hz, 1H), 7.77 (d, J = 8.5 Hz, 1H),
.60 (dd, J = 8.5, 2.6 Hz, 1H), 7.40 (d, J = 12.6 Hz, 1H), 7.21 (d, J = 12.3
8
7
Step 2. 1-[(2R)-1,4-Dioxan-2-yl]-N-methylmethanamine hydro-
chloride(5.37g, 32.1mmol),N-[1-{[(tert-butoxycarbonyl)amino]sulfonyl}-
pyridin-4(1H)-ylidene]-N-methylmethanaminium (9.7 g, 32.1 mmol), and
triethylamine (5.4 mL, 39 mmol) were slurried in DCM (200 mL) and
stirred at ambient temperature. After 4 h, the solution was concentrated in
vacuo and purified by flash column chromatography (0ꢀ75% EtOAc/
hexanes gradient) to afford 8.81 g of tert-butyl {[[(2R)-1,4-dioxan-
Hz, 1H), 2.72 (s, 6H). LRMS (ESI) calcd for (C16
H] 364.0; found 364.1.
3 3
H15ClN O S) [M þ
þ
Step 2. A flask was charged with compound 13a (1.22 g, 3.35 mmol),
-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan)-1H-pyrazole (2.09 g,
0.1 mmol), tri-t-butylphoshonium tetrafluoroborate (97.0 mg,
1
1
0
1
.334 mmol), Pd
2
(dba)
3
(154 mg, 0.168 mmol), KF (643 mg, 11.1
2-ylmethyl](methyl)amino]sulfonyl}carbamate (88%). H NMR (600
mmol), and DMF (40 mL). The mixture was sparged with argon for
0 min and then heated to 100 ꢀC for 2 h. The reaction mixture was
cooled to ambient temperature and then diluted in EtOAc (500 mL) and
washed with H
O (2 ꢁ 100 mL). The organic layer was separated and
dried over MgSO . The solution was concentrated in vacuo to afford a
MHz, DMSO-d
6
) δ 10.93 (s, 1H), 3.72ꢀ3.68 (m, 1H), 3.66ꢀ3.63
1
(m, 2H), 3.62ꢀ3.58 (m, 1H), 3.55ꢀ3.50 (m, 1H), 3.44ꢀ3.39 (m, 1H),
3.21ꢀ3.14 (m, 3H), 2.81 (s, 3H), 1.39 (s, 9H). LRMS (ESI) calcd for
þ
(C11
H
22
N
2
O
6
SNa) [M þ Na] 333.1; found 333.1.
2
Step 3. tert-Butyl {[[(2R)-1,4-dioxan-2-ylmethyl](methyl)amino]
sulfonyl}carbamate (8.81 g, 28.4 mmol) was dissolved in DCM
(100 mL) and trifluoroacetic acid (100 mL) and stirred at ambient
temperature. After 6 h, the solution was concentrated and azeotroped
twice with heptane to afford 8.35 g of {[((2R)1,4-dioxan-2-ylmethyl)-
4
yellow solid. The yellow solid was recrystallized from 1:4:4 MeOH/
1
DCM/hexanes to afford 1.01 g of 59 (74%). H NMR (600 MHz,
6
DMSO-d ) δ 10.52 (s, 1H), 9.20 (d, J = 2.4 Hz, 1H), 8.57 (d, J = 1.8 Hz,
1H), 8.47 (s, 1H), 8.14 (s, 1H), 8.00 (d, J = 2.4 Hz, 1H), 7.75 (d, J = 8.4
1
Hz, 1H), 7.57 (dd, J = 8.4, 2.4 Hz, 1H), 7.32 (d, J = 12.6 Hz, 1H), 7.23 (d,
(methyl)amino]sulfonyl}ammonium trifluoroacetate (91%). H NMR
J = 12.0 Hz, 1H), 3.88 (s, 3H), 2.73 (s, 6H). LRMS (ESI) calcd for
(600 MHz, DMSO-d
6
) δ 6.70 (s, 2H), 3.72ꢀ3.63 (m, 3H), 3.62ꢀ3.58
þ
(C
20
H
20
N
5
O
Benzyl [(2R)-1,4-Dioxan-2-ylmethyl]methylcarbamate.
3
S) [M þ H] 410.1; found 410.1.
(m, 1H), 3.56ꢀ3.50 (m, 1H), 3.44ꢀ3.39 (m, 1H), 3.21ꢀ3.16 (m, 1H),
2.90ꢀ2.86 (m, 2H), 2.65 (s, 3H). LRMS (ESI) calcd for (C
6 15 2 4
H N O S)
þ
Step 1. 1-(1,4-Dioxan-2-yl)-N-methylmethanamine (10.2 g, 78.0
mmol) was dissolved in 200 mL of DCM. Benzyl chloridocarbonate
[M þ H] 211.1; found 211.1.
0
N-[(2R)-1,4-Dioxan-2-ylmethyl]-N-methyl-N -[3-(1-methyl-
1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]
(
16.4 mL, 117 mmol) and triethylamine (32.6 mL, 234 mmol) were
55
added, and the solution was stirred at ambient temperature. After 12
h, the solution was concentrated and then diluted with EtOAc (1 L)
and washed with saturated aqueous sodium bicarbonate (100 mL)
pyridin-7-yl]sulfuric Diamide (81). Step 1. Compound 7 (9.1 g,
28.3 mmol), {[((2R)1,4-dioxan-2-ylmethyl)(methyl)amino]sulfonyl}-
ammonium trifluoroacetate (8.35 g, 25.8 mmol), Pd
2 3
dba (1.18 g, 1.29
and H
2
O (100 mL). The organic layer was separated, dried with
mmol), Xantphos (2.24 g, 3.87 mmol), Cs CO (33.6 g, 103 mmol), and
2
3
magnesium sulfate, filtered, concentrated in vacuo, and purified by
flash column chromatography (0ꢀ100% EtOAc/hexanes gradient)
to afford 19.0 g of racemic benzyl (1,4-dioxan-2-ylmethyl)methyl-
carbamate (92% yield).
1,4-dioxane (300 mL) were combined in a dry flask. Argon was sparged
through the mixture for 5 min, and then the mixture was heated to 95 ꢀC.
After 3 h, the solution was concentrated in vacuo, diluted with EtOAc
2
(1.5 L), and washedwithH O (500 mL) and brine (500 mL). The organic
The racemic mixture was dissolved in 75 mL of heptane and 25 mL of
isopropyl alcohol. The material was resolved on a chiral AD column
layer was isolated, dried with magnesium sulfate, filtered, concentrated in
vacuo, and purified by flash column chromatography (0ꢀ100% EtOAc/
0
(
9
15% isopropyl alcohol/heptane) to afford 8.4 g of enantiomer A [τ
.35 min (analytical chiral HPLC, AD column, 0.46 cm ꢁ 25 cm id, 15%
isopropyl alcohol/heptane, isocratic, flow rate = 0.75 mL/min)] and
.4 g of enantiomer B [τ : 10.92 min (analytical chiral HPLC, AD
R
:
hexanes gradient) to afford 9.95 g of N -(3-chloro-5-oxo-5H-benzo-
[4,5]cyclohepta[1,2-b]pyridin-7-yl)-N-[(2R)-1,4-dioxan-2-ylmethyl]-
1
N-methylsulfuric diamide (86%). H NMR (600 MHz, DMSO-d
6
) δ
8
R
10.57 (s, 1H), 8.97 (d, J = 2.4 Hz, 1H), 8.49 (d, J = 2.4 Hz, 1H), 7.96
(d, J = 2.4 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.57 (dd, J = 8.4, 2.4 Hz, 1H),
7.41 (d, J = 12.6 Hz, 1H), 7.22 (d, J = 12.6 Hz, 1H), 3.65ꢀ3.60 (m, 2H),
3.59ꢀ3.52 (m, 2H), 3.45ꢀ3.40 (m, 1H), 3.38ꢀ3.33 (m, 1H), 3.15ꢀ3.10
column, 0.46 cm ꢁ 25 cm id, 15% isopropyl alcohol/heptane, isocratic,
flow rate = 0.75 mL/min)].
1
H NMR (600 MHz, DMSO-d
6
) δ 7.36ꢀ7.32 (m, 4H), 7.30ꢀ7.27
(
3
(
m, 1H), 5.03 (s, 2H), 3.72ꢀ3.56 (m, 4H), 3.54ꢀ3.46 (m, 1H),
(m, 3H), 2.77 (s, 3H). LRMS (ESI) calcd for (C20
H
21ClN
3
O
5
S)
þ
.44ꢀ3.38 (m, 1H), 3.26ꢀ3.10 (m, 3H), 2.90ꢀ2.84 (m, 3H). LRMS
[M þ H] 450.1; found 450.1.
þ
0
ESI) calcd for (C H NO ) [M þ H] 266.1; found 266.2.
Step 2. N -(3-Chloro-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-
7-yl)-N-[(2R)-1,4-dioxan-2-ylmethyl]-N-methylsulfuric diamide (8.05 g,
17.9 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
1H-pyrazole (11.2 g, 53.7 mmol), Pd (dba) (0.820 g, 0.895 mmol),
14
20
4
The absolute stereochemistry of enantiomer A and B was determined
by comparison of HPLC retention times to benzyl [(2S)-1,4-dioxan-2-
ylmethyl]methylcarbamate, which was prepared from commercially
available (2R)-3-(benzyloxy)propane-1,2-diol as described in the Sup-
porting Information.
N-[(2R)-1,4-Dioxan-2-ylmethyl]-N-methylsulfuric Diamide.
Step 1. Benzyl [(2R)-1,4-dioxan-2-ylmethyl]methylcarbamate (enantio-
mer A, 8.4 g, 31.7 mmol) was dissolved in dry ethanol (200 mL) under a
nitrogen atmosphere, and then 10% (w/w) palladium on carbon (0.84 g,
2
3
t-Bu PHBF (0.520 g, 1.79 mmol), potassium fluoride (3.43 g, 59.1
3
4
mmol), and DMF (200 mL) were combined in a dry flask. The mixture
was sparged with argon for 5 min and then heated to 100 ꢀC. After 3 h, the
solution was diluted with EtOAc and washed with saturated sodium
bicarbonate, H O, and brine. The organic layer was dried with magnesium
2
sulfate, filtered, and concentrated in vacuo. The residue was crystallized
from a mixture of methanol (70 mL), DCM (275 mL), and hexanes
0.79 mmol) and 10 N HCl (6.4 mL) were added. The flask was placed
4
105
dx.doi.org/10.1021/jm200112k |J. Med. Chem. 2011, 54, 4092–4108

Journal of Medicinal Chemistry
ARTICLE
(
600 mL) to afford 6.17 g of N-[(2R)-1,4-dioxan-2-ylmethyl]-N-methyl-
Accession Codes
PDB ID 3R7O.
0
N -[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]-
pyridin-7-yl]sulfuric diamide (70%). H NMR (600 MHz, DMSO-d
1
1
1
6
) δ
0.53 (s, 1H), 9.19 (d, J = 1.8 Hz, 1H), 8.56 (d, J = 2.4 Hz, 1H), 8.46 (s,
H), 8.13 (s, 1H), 7.96 (d, J = 2.4 Hz, 1H), 7.75 (d, J = 9.0 Hz, 1H), 7.55
’
AUTHOR INFORMATION
Corresponding Author
*
(dd, J= 8.4, 2.4 Hz, 1H), 7.32 (d, J= 12.6 Hz, 1H), 7.23 (d, J= 12.6 Hz, 1H),
Phone: 617-992-2057. E-mail: jason_katz2@merck.com. Fax:
3
3
.88 (s, 3H), 3.65ꢀ3.60 (m, 2H), 3.59ꢀ3.51 (m 2H), 3.45ꢀ3.40 (m, 1H),
.38ꢀ3.33 (m, 1H), 3.15ꢀ3.10 (m, 3H), 2.78 (s, 3H). LRMS (ESI) calcd
617-992-2403.
þ
for (C24
H
26
N
5
O
5
S) [M þ H] 496.2; found 496.1.
In Vitro Kinase Assays. As described in ref 31, c-Met-catalyzed
phosphorylation of N-biotinylated peptide (EQEDEPEGDYFEWLE-
’
ACKNOWLEDGMENT
CONH
2
) was measured using a time-resolved fluorescence resonance
We thank Bruce Adams for assistance in NMR structural
energy transfer assay. To evaluate kinase selectivity, a single concentra-
tion (1 μmol/L) of 81 was tested using 216 kinases by Upstate
Biotechnology, Inc. (Millipore), which also determined the IC50 for
DRAK1, DYRK2, IRAK1, IRAK4, MELK, and MLK1. The IC50 for Ron,
Mer, Flt1, Flt3, Flt4, KDR, PDGFRβ, FGFR1, FGFR2, FGFR3, TrkA,
and TrkB were determined using time-resolved fluorescence resonance
energy transfer assays similar to the c-Met kinase assay.
analysis and Regina Black for purification of key intermediates. In
addition, we thank Elizabeth Stanton for analysis and compila-
tion of analytical data.
’
ABBREVIATIONS USED
0
ATP, adenosine triphosphate; BINAP, 2,2 -bis(diphenylphos-
0
phino)-1,1 -binaphthyl; dba, dibenzylideneacetone; DCM, dichloro-
Cellular Assays. As described in ref 31, proliferation and viability of
tumor cells was measured using the ViaLight PLUS kit (Cambrex).
Analysis of phosphorylation status of c-Met, in cells: Tumor cells were
treated for 2 h with 81 or vehicle in RPMI 1640 supplemented with 10%
fetal bovine serum and 10 mmol/L HEPES. When called for, the cells
were stimulated with HGF or EGF during the last 10 min of the 2 h
incubation. The cells were lysed with a denaturing or nondenaturing
buffer containing phosphatase and protease inhibitors and subjected to
Western blot or immunoprecipitationꢀWestern blot analysis.
methane; DMF, N,N-dimethylformamide; DMSO, dimethylsulfox-
0
ide; dppf, 1,1 -bis(diphenylphosphino)ferrocene; EtOAc, ethyl
acetate; HGF, hepatocyte growth factor; HGFR, hepatocyte
growth factor receptor; IP, intraperitoneal injection; iv, intrave-
nous; Ms, methanesulfonyl; NMP, N-methylpyrrolidinone; PK,
pharmacokinetic; PPA, polyphosphoric acid; SAR, structureꢀ
activity relationship; TBAF, tetrabutylammonium fluoride;
Tf, trifluoromethanesulfonyl; TFA, trifluoroacetic acid; THF,
tetrahydrofuran; Xantphos, 4,5-bis(diphenylphosphino)-9,9-
dimethylxanthene
Tumor Xenograft Models. As described in ref 31, GTL-16 cells
were inoculated sc into the flank of female nude CD-1 nu/nu mice.
When mean tumor size reached a predetermined range, the mice were
randomized and given vehicle or 81 by po gavage once or twice daily.
Tumor volumes were determined using calipers. The percentage
increase in the volume of a xenograft tumor on day n versus day 0
’
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’
ASSOCIATED CONTENT
1
S
Supporting Information. Full experimentals and H
b
NMR data are available for compounds 3bꢀe, 10ꢀ11, 21ꢀ29,
3
0ꢀ33, 35ꢀ36, 38ꢀ44, 46ꢀ58, 60ꢀ80, and 82 and structure-
determination statistics for the crystal structure of the c-Met
kinase domain complex with 82 are presented. This material is
available free of charge via the Internet at http://pubs.acs.org.
4
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Journal of Medicinal Chemistry
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46) Measured at Essen Biosciences in Chinese hamster lung cells
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47) Methanesulfonamide 47 has a calculated (ACD laboratories)
= 8.87 ( 0.20, while 57 has a calculated pK = 4.23 ( 0.20.
48) Identified by incubating the compound with rat and human
hepatocytes and analysis of metabolites via HPLC and mass spectrometry.
49) Monomethyl 71 has been identified as a major metabolite of 59
in rat hepatocytes.
50) Rickert, K. W.; Patel, S. B.; Allison, T. J.; Byrne, N. J.; Darke,
(
(
pK
a
a
(
(
(
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51) As predicted by density-functional theory (DFT) geometry
calculations at the B3LYP/6-31 g* level.
52) Berthou, S.; Aebersold, D. M.; Schmidt, L. S.; Stroka, D.; Heigl,
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(
4
108
dx.doi.org/10.1021/jm200112k |J. Med. Chem. 2011, 54, 4092–4108