Discovery and structure-activity relationship of 3-aminopyrid-2-ones as potent and selective interleukin-2 inducible T-cell kinase (Itk) inhibitors

Article abstract of DOI:10.1021/jm101499u

Interleukin-2 inducible T-cell kinase (Itk) plays a role in T-cell functions, and its inhibition potentially represents an attractive intervention point to treat autoimmune and allergic diseases. Herein we describe the discovery of a series of potent and

Full text of DOI:10.1021/jm101499u

ARTICLE
pubs.acs.org/jmc
Discovery and Structure-Activity Relationship
of 3-Aminopyrid-2-ones as Potent and Selective
Interleukin-2 Inducible T-Cell Kinase (Itk) Inhibitors†
Jean-Damien Charrier,*,‡ Andrew Miller,‡ David P. Kay,‡ Guy Brenchley,‡ Heather C. Twin,‡
Philip N. Collier,‡ Sharn Ramaya,‡ Shazia B. Keily,‡ Steven J. Durrant,‡ Ronald M. A. Knegtel,‡
Adam J. Tanner,§ Kieron Brown,§ Adam P. Curnock,§ and Juan-Miguel Jimenez‡
‡Department of Chemistry and §Department of Biology, Vertex Pharmaceuticals (Europe) Ltd., 88 Milton Park, Abingdon,
Oxfordshire OX14 4RY, U.K.
S Supporting Information
b
ABSTRACT: Interleukin-2 inducible T-cell kinase (Itk) plays a
role in T-cell functions, and its inhibition potentially represents
an attractive intervention point to treat autoimmune and
allergic diseases. Herein we describe the discovery of a series
of potent and selective novel inhibitors of Itk. These inhibitors
were identified by structure-based design, starting from a
fragment generated de novo, the 3-aminopyrid-2-one motif.
Functionalization of the 3-amino group enabled rapid enhance-
ment of the inhibitory activity against Itk, while introduction of
a substituted heteroaromatic ring in position 5 of the pyridone
fragment was key to achieving optimal selectivity over related kinases. A careful analysis of the hydration patterns in the kinase active
site was necessary to fully explain the observed selectivity profile. The best molecule prepared in this optimization campaign, 7v,
inhibits Itk with a Ki of 7 nM and has a good selectivity profile across kinases.
’ INTRODUCTION
pharmaceutical companies to initiate medicinal chemistry programs
in order to identify small molecules Itk inhibitors.9 BMS and BI have
recently disclosed a series of 2-aminothiazoles10,11 (e.g., 1 (BMS-
509744)) and benzimidazoles12-16 (e.g., 2) as potent and selective
Itk inhibitors (Figure 1). Compounds from these two scaffold families
have been shown to be effective at inhibiting IL-217 and IL-4
production and lung inflammation in mice.18
An HTS campaign of our corporate compound collection led to
the identification of potent Itk inhibitors from distinct scaffold series.
However, these series showed cross-reactivity with a large propor-
tion of our kinase counterscreening panel, and initial attempts to
modify their selectivity profiles proved futile. Rather than deconvo-
luting these scaffolds back to the features required for their binding
to Itk and then rebuilding potency/selectivity, we hypothesized that
the likelihood of success would be enhanced by starting from a
druglike, easy to derivatize hinge binding fragment.
In this report, we outline our efforts to identify a novel series of
potent and selective Itk inhibitors from de novo design. The
3-aminopyrid-2-one fragment was selected as a core scaffold because
of its potential ability to form a multiple hydrogen bond network
with the hinge region of the ATP binding site of Itk similar to
maleimides such as staurosporine and the CDK2 natural inhibitor
hymenialdisine.19-21 Compound 7v represents the best molecule
The five Tec family tyrosine kinase members, Btk, Bmx, Itk, Rlk,
and Tec, are expressed primarily in hematopoetic cells and have
important roles in the development and function of leucocytes
including B-cells, T-cells, and mast cells.1 Three members of the Tec
family, Itk, Rlk and Tec, are activated following antigen receptor
engagement in T-cells and transmit signals to downstream effectors,
including PLC-γ. A fourth member, Btk, appears to act indepen-
dently of T-cell signaling and is essential for B-cell development and
activation. A biological role for the final member of this family, Bmx,
is not yet clearly elucidated.
Itk, a key member of this family, is predominantly expressed in
T-cells, and a number of factors point to the importance of this kinase
in immune diseases. Deletion of Itk in mice results in reduced T-cell
receptor (TCR) induced proliferation and secretion of the cytokines
IL-2, IL-4, IL-5, IL-10, and IFN-γ.2-5 In Itk-/- mice, the im-
munological symptoms of allergic asthma are attenuated and lung
inflammation, eosinophil infiltration, and mucous production are
drastically reduced in response to challenge with the allergen
ovalbumin.6 Itk has also been implicated in atopic dermatitis. This
gene has been reported to be more highly expressed in peripheral
blood T-cells from patients with moderate and/or severe atopic
dermatitis than in controls or patients with mild atopic dermatitis.7
This wealth of information on the role of Itk in T-cell function
provides strong rationale for considering inhibition of this target to
treat autoimmune and allergic diseases8 and has prompted many
Received: November 22, 2010
Published: March 10, 2011
r
2011 American Chemical Society
2341
dx.doi.org/10.1021/jm101499u J. Med. Chem. 2011, 54, 2341–2350
|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. Structures of Itk inhibitors reported by Bristol Myers Squibb and Boehringer Ingelheim.
prepared in the pyridone series and shows inhibitory activity against
Itk (Kiapp of 7 nM for Itk) comparable with compounds reported in
the literature (e.g., 1 or 2) and has a good selectivity profile.
’ CHEMISTRY
In order to rapidly optimize potency, selectivity, and physico-
chemical properties in the 3-aminopyrid-2-ones series, a syn-
thetic route was required that would allow changes to the amide
portion (R group) and C-5 (hetero)aryl (R0 group) in a flexible
and efficient manner (Figure 2).
Figure 2. Generic structure for 3-aminopyrid-2-one series.
bond is formed with the hinge. Unexpectedly, the aromatic CH in
the ortho-position of the benzamide moiety also seems to provide
additional hydrogen bonding in the hinge region, as the X-ray
reveals a distance of 3.9 Å with the Met438 backbone CdO. In
addition to these contacts with the hinge, the pyridine ring disrupts a
salt bridge formed between Lys391 and Asp500 that is observed in
the Itk apo structure24 and the aromatic nitrogen of the inhibitor
rests within H-bond distance (3.5 Å) of the salt-bridge lysine 391.
The pyridine ring itself is able to make an edge-to-face π-σ
interaction with Phe435 located at the back of the active site.
Analysis of this initial crystal structure also suggests that further
hydrophobic interactions with residues Phe437 and Leu379, located
above the hinge at the entrance of the active site, could be made from
appropriate substitution on the benzamide ring. This ring, by virtue
of its proximity to bulk solvent, would also be a suitable attachment
point to allow introduction of solubilizing groups (Figure 4b).
Additional affinity and selectivity over other kinases might also
be achieved by variation of the pyridine ring: alternative hetero-
aryls with appropriate subsitution are likely to alter interaction
with the “gatekeeper” residue Phe435, which, though not unique
to Itk, is not strongly conserved across kinases.
The general synthetic route that was developed is illustrated with
the preparation of compound 7o in Scheme 1.22 The key boronate
intermediate 4 is prepared from 3 using pinacoldiboronate in the
presence of Pd as a catalyst.23 Suzuki coupling with the appropriate
heteroaryl halide partner affords 5, which under Cbz deprotection
and final acylation provides the final compound 7o. The preparation
of compound 7o was performed in a highly efficient four-step
sequence with an overall yield of 28% starting from easily accessible
building blocks.
Compounds 7a-y were all prepared using this highly versatile
sequence.
Compound 11, in which the amide functional group is
connected to the pyridone through the carbonyl, is prepared as
described in Scheme 2. Malondialdehyde 8 reacts with cyanoa-
cetamide to generate intermediate 9, which is further hydrolyzed
into compound 10. Amide coupling with the suitably functiona-
lized aniline provides the final compound 11.19
’ RESULTS AND DISCUSSION
To assess the potential of our de novo fragment approach, the
commercially available vasodilator amrinone 12 was envisioned to be
an excellent druglike starting point, as it combines a hinge binding
3-aminopyrid-2-one motif and offers a readily exploitable functiona-
lization point to rapidly introduce diversity (amino group at position
3 of pyridone nucleus). A simple benzoyl capping of the exocyclic
amine of fragment amrinone yielded the benzamide analogue 7a, with
micromolar potency against Itk and good ligand efficiency (20.2; LE =
pKi/MW) (Figure 3). As a hit compound, 7a is likely to require
100- to 1000-fold potency improvement against Itk to become
comparable to reported inhibitors in the literature (e.g., 1 and 2).
An X-ray structure of compound 7a (PDB code 3QGW) bound
into the ATP binding site of Itk was determined with a resolution of
2.1 Å (Figure 4). This structure confirms the involvement of the
aminopyridone binding motif in a network of hydrogen bonds with
the hinge: the distance between the Glu436 backbone CdOandthe
endocyclic N-H is measured to be 2.7 Å, and the distance between
the Met438 backbone N-H and the pyridone CdO is 2.3 Å. The
longer distance of 4.0 Å between the Met438 backbone CdO and
inhibitor exocyclic N-H suggests that only a weak third hydrogen
With the objective of transforming hit compound 7a (Ki =1.3μM)
into an inhibitor that would be more suitable for lead optimization, we
initially examined variations of the amide, targeting the additional
lipophilic contacts in this area of the active site (Table 1). Progress was
monitored using inhibitory activity against Itk (Ki) and ligand
efficiency (LE = pKi/MW).
Reduction of the aromatic benzamide (compound 7b) or intro-
duction of a spacer group between the aromatic ring and the carbonyl
amide (compound 7c) resulted in a >20-fold loss of potency. These
results are in line with the observation that the aromatic CH in the
ortho-position of the benzamide 7a forms a hydrogen-bond-like
interaction with a hinge carbonyl and can be further rationalized by
the proximity of the phenyl ring to Phe437 which may allow
productive π-π interaction.
Small lipophilic substitution on the benzamide ring was
tolerated, preferably at position 4 (7f, Ki = 1.2 μM, LE = 19.4).
The effect of larger hydrocarbon motifs in the para-position was
therefore examined in a systematic fashion, and a small array of
compounds showed that the inhibitory activity could improve
2342
dx.doi.org/10.1021/jm101499u |J. Med. Chem. 2011, 54, 2341–2350

Journal of Medicinal Chemistry
ARTICLE
Scheme 1a
a Reagents and conditions: (a) PdCl2(PPh3)2, KOAc, dioxane, 77%; (b) Pd(PPh3)4, Na2CO3, toluene/EtOH/water, 67%; (c) H2, Pd/C, MeOH/
AcOEt, 100%; (d) 4-(piperidin-1-yl)benzoic acid, POCl3, MeCN, 55%.
Scheme 2a
a Reagents and conditions: (a) morpholine, AcOH, benzene, 83%; (b) 9
N HCl, reflux, 54% ; (c) 4-(piperidin-1-yl)aniline, EDCI, Et3N,
DMF, 20%.
Figure 4. X-ray structure of compound 7a in the Itk active site (RCSB
Protein Data Bank code 3QGW). Itk crystal structures were visualized
with Pymol, version 1.1r1 (Schro€dinger LLC, NY, 2010). (a) X-ray
structure of compound 7a (green) in Itk active site (gray). Important
residues, including those hydrogen-bonding to the ligand, are high-
lighted in stick. (b) Shape and chemical complementarity between the
Itk active site (surface representation) and compound 7a (green).
Figure 3. From de novo design to hit compound 7a.
with increasing size of the lipophile while maintaining ligand
efficiency. This culminated with a cyclohexyl group for which
potency started to plateau and ligand efficiency to decrease (7j,
Ki = 0.22 μM, LE = 17.8). This saturated six-membered ring was
further modified and could be advantageously replaced with a
piperidine ring (7m, Ki = 0.093 μM, LE = 18.8).
Reversing the amide linker between the pyridone core and the
phenyl ring showed similar inhibitory activity (11, Ki = 0.14 μM
vs 7m, Ki = 0.093 μM), in agreement with our earlier observation
2343
dx.doi.org/10.1021/jm101499u |J. Med. Chem. 2011, 54, 2341–2350

Journal of Medicinal Chemistry
ARTICLE
Table 1. Structures and Inhibitory Activity of Variants 7a-m
and 7k: KiKit = 27 and 46 nM, respectively; KiFlt3 = 11 and 8 nM,
respectively; KiSrc = 1.32 and 2.8 μM, respectively). Starting from
compound 7m, we therefore initiated a second phase of the lead
optimization campaign based on the hypothesis that selectivity
might be modulated if interactions with residues that are deeply
buried in the active site were altered (e.g., “gatekeeper” residue
Phe435 and Asp500/Lys391 salt bridge residues).
and 11a
Both residues Phe435 and Asp500 can potentially be accessed
from substitution on the pyridine nucleus in 7m; hence, we
examined the effect of a methylamino substitution in position 2
of the pyridine nucleus (Table 2). Compound 7n showed a 13-
fold enhancement in potency (Ki = 7 nM) and improved ligand
efficiency (LE = 20.2). Replacement of the 2-methylaminopyr-
idine moiety with a 2-methylaminopyrimidine (compounds 7o)
led to a similar profile, in terms of both potency and selectivity.
An X-ray structure of 7o (PDB code 3QGY) was determined with
a resolution of 2.1 Å in order to understand the increase in affinity
observed (Figure 5). Although originally expected to improve
potency by introducing a new hydrogen bond to the salt-bridge
Asp500, instead the methylamino group was found to promote
conformational changes in nearby residues, resulting in the creation of
a new lipophilic pocket. Changes include a small 10° rotation of the
phenyl ring of Phe435, a major 160° rotation of the ε-methyl group of
Met410, and the formation of a new hydrogen bond with Glu406 of
the C-helix (Figure 5a). The methyl group ends up deeply buried in
the newly formed pocket lined by the phenyl ring of Phe435 above
and the side chains of Met410 and Val419 in the back and below,
respectively (Figure 5b). The conformations of residues within van
der Waals range of the aminomethylheteroaryl moiety were found to
be conserved in subsequent crystal structures, independent of the
nature of the heteroaryl ring (results not shown). Because of the
vicinity of the methylamino substituent to the Glu406 carboxylate in
the crystal structure (Figure 5), the hydrogen bond donating ability of
the amino linker was considered to be key to the observed improve-
ment in potency. Indeed, its replacement with a more lipophilic
methanethiol group lost approximately 10-fold in affinity (results not
shown). Given this SAR and the local environment observed in the
crystal structure of 7o, no further hydrophobic substitutions were
attempted at this position. Attempts to further optimize interactions
in this pocket using larger aminoalkyl groups resulted in a significant
reduction in activity (7r vs 7s, Ki = 0.112 μM and Ki > 2 μM,
respectively), in agreement with the space available in the crystal
structure and the rigidity of the Phe435 gatekeeper side chain that
restricts the size of the pocket. No such induced-fiteffects are required
inthe case of Src kinase where the methylamino group canfit into the
existing Src-selectivity pocket; the addition of a methylamino group
therefore also leads to a potency improvement in the case of Src. The
local environment near the methylamino group in Kit and Flt3 is
more challenging to interpret because, in contrast to Itk, several
distinct inactive structures have been published and it is not clear to
which conformation our inhibitors bind.25-28
a Functionalization is at position 3. The asterisk (/) indicates that the
upper concentration of compound was limited by solubility in assay
(15% inhibition at 3 μM).
In addition to the gain in potency via the methylamino group,
the X-ray structure also reveals that the pyrimidine nitrogen is
still 3.5 Å from Lys391, hence able to form a productive H-bond.
This observation is in line with the significant potency reduction
observed with compounds lacking the ring nitrogen acceptor in
this position (7n vs 7q, Ki = 7 and 145 nM, respectively).
In our investigation of the impact of variation of the heteroaryl at
C5, the best results were obtained with compounds 7t and 7u, which
retained good activity against Itk (Ki = 11 and 23 nM, respectively),
in combination with an improved selectivity window with related
that this NH does not make a strong hydrogen bond with
Met438.
It was found empirically through our counterscreen panel of ∼20
diverses enzymes that compounds 7a-m also inhibited a number of
unrelated kinases (e.g., c-Kit, Flt3, and Src), and it became apparent
that modification of the substitution pattern on the benzamide
moiety did not significantly improve their selectivity profile (e.g., 7i
2344
dx.doi.org/10.1021/jm101499u |J. Med. Chem. 2011, 54, 2341–2350

Journal of Medicinal Chemistry
ARTICLE
Table 2. Structures and Inhibitory Activity of Amide Variants 7m-ua
a Functionalization is at position 5.
kinases (7t c-Kit Ki = 155 nM; Flt3 Ki = 110 nM; Src Ki = 590 nM;
7u c-Kit Ki = 270 nM; Flt3 Ki = 1045 nM; Src Ki = 590 nM).
It is difficult to explain the data obtained for compounds 7t and 7u,
since the aromatic nitrogens do not appear to make direct contact
with residues in the active site. Commonly used computational
methods failed to explain these results on the basis of crystallographic
data, since moving a single aromatic nitrogen was predicted to lose the
H-bond with Lys391 (or the equivalent catalytic lysine in other
kinases), and as a result, a reduction in affinity was anticipated.
This expectation was realized for Flt3, Kit, and Src (7t vs 7o)
where potency was reduced by 5- to 20-fold. However, in Itk this
prediction did not hold true and binding affinities of 7t and 7o were
very similar.
Given that direct protein-ligand contacts could not be used to
provide a satisfactory explanation of the observed selectivity pattern,
the possible role of solvation/desolvation was investigated.29 Using
the molecular dynamics tool Watermap, hydration sites in apo-Itk
have been identified.30 One particular hydration site, present in Itk
and absent in Src, Flt3, and Kit, is found close to the position of the
aromatic nitrogen in 7t and 7u. This hydration site in apo-Itk is
predicted to be highly thermodynamically unfavorable; displacement
of this water molecule with a suitable inhibitor could therefore provide
an energy gain, resulting in improved potency against Itk but not
against other kinases that lack this hydration site. The average
orientation of water molecules at this hydration site suggests that
an sp2-nitrogen restores complementarity with the local solvent
structure, in contrast to the corresponding CH analogue. This
hypothesis is also consistent with the SAR of inhibitors 7t and 7r;
7t possesses an sp2-nitrogen which is complementary with solvent
(Ki7t = 11 nM) while 7r does not, leading to a 10-fold loss in potency
(Ki7r = 112 nM).
In the case of compounds 7t and 7u, the loss of H-bond with
Lys391 and the gain in potency through improved complemen-
tarity with the solvent structure in the Itk active site cancel each
other, resulting in similar binding affinity to 7n and 7o.
To further explore this unique feature in the context of
compounds with druglike solubility and cell permeability, a
range of inhibitors with the solubilizing motif pyrrolidino-
methylpyrrolidine were prepared (7v-y). The pyrrolidino-
methylpyrrolidine moiety was identified by virtual screening of
a library of commercially available amines against the Itk crystal
structure, using Glide with SP scoring (Schr€odinger LLC, NY,
2010). Inhibitors 7v-y possess diverse H-bond acceptor fea-
tures able to displace the Itk-specific water molecule (Table 3).
2345
dx.doi.org/10.1021/jm101499u |J. Med. Chem. 2011, 54, 2341–2350

Journal of Medicinal Chemistry
ARTICLE
bulk solvent, nearby water molecules will orient their dipoles accord-
ingly and project their own oxygen atoms toward subsequent solvation
shells. The cyano nitrogen of 7y could mimic the oxygen atoms of
water molecules in the second solvation shell of the original hydration
site, thus conserving the optimal solvent structure in the Itk active site.
In addition to retaining good potency and selectivity, com-
pounds 7v-y have been designed for their druglike properties
(Table 3); all have solubility, lipophilicity, and permeability
profiles thatare consistentwithgoodcell penetration. Ofparticular
interest, compound 7v shows good Itk affinity, selectivity, and
physicochemical properties consistent with oral druglike charac-
teristics(solubilityatpH7.4 is 150 μg/mL;goodpermeabilitywith
no major efflux as measured in Caco2 study, A-B 10.8 ꢀ 10-6 cm/s,
B-A 32.4.10-6 cm/s) (Tables 3 and 4).
The best molecule prepared in this rapid optimization campaign,
compound 7v, has inhibitory activity against Itk comparable with
compounds reported in the literature (e.g., BMS-509744)9 and has a
good selectivity profile across a representative panel of kinases. The
kinases used to establish the extended selectivity profile of 7v listed in
Table 4 provide representative examples of kinases from all major
groups of enzymatically active kinases as defined by Manning et al.32
(assay data obtained using the Kinase Profiler service provided by
Millipore, Billerica, MA, U.S.). Additionally, physicochemical proper-
ties of compound 7v are consistent with good cell penetration.
Compound 7v therefore represents a good chemical probe for further
mechanistic and structural investigations into the Itk pathway, results
of which will be presented elsewhere.
’ CONCLUSION
A novel class of Itk inhibitors based on the 3-aminopyrid-2-
one fragment was identified. Structure based design helped build
in potency and rationalize the subtleties of the Itk SAR. Careful
analysis of the hydration patterns in the kinase active sites helped
to explain the selectivity that was observed and may prove to be a
useful tool to engineer selectivity in future projects.
The discovery of compound 7v illustrates the fact that a
fragment-like de novo starting point can rapidly evolve into
compounds with good potency and selectivity profile.
Figure 5. X-ray structure of compound 7o in Itk active site (RCSB
Protein Data Bank code 3QGY). (a) X-ray structure of compound 7o
(green) in Itk active site (purple). Important residues, including those
involved in conformational changes, are highlighted as sticks. These
conformational changes from the apo-enzyme (white) to the complex
with 7o (purple) are highlighted with orange arrows. Movements of
Phe435 (small arrow) and Met410 (large arrow) enable the formation of
a new lipophilic pocket. (b) X-ray structure of compound 7o (green) in
ATP binding site (surface representation). The methylamino group of
7o fills a newly created pocket lined by Met410, Phe435, and Val419.
’ MATERIALS AND METHODS
General Experimental Details. All commercially available sol-
vents and reagents were used as received. Microwave reactions were
carried out using a CEM Discovery microwave. Analytical thin layer
chromatography was carried out using glass-backed plates coated with
Merck Kieselgel 60 GF240. Plates were visualized using UV light (254 or
366 nm) and/or by staining with potassium permanganate followed by
heating. Flash chromatography was carried out on an ISCO Combiflash
Companion system, eluting with a 0-100% EtOAc/petroleum ether
As expected, pyrimidine 7v and pyridine 7w had similar profiles
to compounds 7t and 7u described above.
gradient. Samples were applied preabsorbed on silica. 1H NMR and 13
C
Replacement of the aromatic nitrogen H-bond acceptor
in pyridine 7w with an aromatic C-F H-bond acceptor31
resulted in fluorophenyl derivative 7x with retained inhibitory
activity against Itk and good selectivity versus c-Kit, Flt3 and Src.
As anticipated, the profile obtained for 7x was completely
different from that of compound 7q, which cannot rely on the
aromatic C-F (C-H instead) to fill the hydration site unique
to Itk.
The nitrile hydrogen bond acceptor in cyanophenyl derivative 7y is
projected deeper into the active site and no longer overlays with the
Itk-specific hydration site. To explain the retention of the selectivity
profile, we hypothesize that the second solvation shell around the
unique Itk hydration site may play a role. As the Itk-specific hydration
site projects the oxygen atoms of occupying water molecules toward
NMR spectra were recorded at 400 MHz using a Bruker DPX 400
instrument. HRMS spectra were recorded on a Waters Micromass Q Tof
Premier spectrometer, operating in positive electrospray mode. MS
samples were analyzed on a MicroMass Quattro Micro mass spectro-
meter operated in single MS mode with electrospray ionization. Samples
were introduced into the mass spectrometer using chromatography.
Infrared spectra were recorded in the range 4000-600 cm-1 using a
Nicolet Avatar 360 FTIR spectrometer and were recorded neat.
Elemental analysis was carried out on a Control Equipment Corporation
440 elemental analyzer. Melting points were measured on a B€uchi B-545
electrothermal digital melting point apparatus and are uncorrected. All
final products had a purity of g95%. The purity of final products was
determined by either combustion analysis or HPLC. Combustion
analyses were performed on a Control Equipment Corporation 440
2346
dx.doi.org/10.1021/jm101499u |J. Med. Chem. 2011, 54, 2341–2350

Journal of Medicinal Chemistry
ARTICLE
Table 3. Structures, Selectivity Profiles, and Physicochemical Properties of Analogues 7v-y
Table 4. Extended Selectivity Profile of Compound 7v
elemental analyzer. HPLC purity was measured on an Agilent HP1100
HPLC instrument with a Waters Quattro MS system equipped with an
ACE C8 reverse phase column (4.6 mm ꢀ 150 mm, 5 μm). The mobile
phases were acetonitrile/methanol (1:1) and water (20 mM, pH 7.0,
Tris phosphate).
3-Benzyloxycarbonylamino-5-(4,4,5,5-tetramethyl-1,3,2-diox-
aborolan-2-yl)-(1H)-pyridin-2-one (4)22. A mixture of 3-benzylox-
ycarbonylamino-5-iodo-(1H)-pyridin-2-one 3 (100 g, 260.5 mmol, 1.0
equiv), bis(pinacolato)diboron (69.4 g, 273.3 mmol, 1.05 equiv), and
potassium acetate (76.64 g, 780.8 mmol, 3.0 equiv) in anhydrous dioxane
(900 mL) was degassed and flushed with nitrogen. PdCl2(PPh3)2 (5.48 g, 7.8
mmol, 3 mol %) was added and the mixture heated to reflux overnight. After
the mixture was cooled to ambient temperature, the solvent was removed in
vacuo, the residue was dissolved in ethyl acetate, washed with aqueous brine,
and dried (MgSO4), and the solvent was removed in vacuo. The crude
product was purified by column chromatography, eluting with 20% ethyl
acetate/petroleum ether to give, after concentration, an orange solid. This was
treated with n-heptane (500 mL) and heated to reflux. On cooling the
resultant precipitate was collected by filtration and washed with hexane to give
4 as a golden solid (77.3 g, 201 mmol, 77%): mp 105.1-106.2 °C; 1H NMR
(CDCl3) δ1.35(12H, s), 4.02(3H, s),5.24(2H, s), 7.20(1H, s), 7.43-7.40
(5H, m), 8.21 (1H, s), 8.65 (1H, s); 13C NMR (CDCl3) δ 25.2, 27.3, 54.2,
67.6, 84.3, 122.5, 128.7, 128.8, 129.0, 130.7, 136.4, 147.1, 153.6, 155.3;
IR 2935, 1719, 1533, 1476, 1438, 1359, 1254, 1216, 1141, 1049, 757, 687;
ES M þ H 385.20, M - H 383.18. Anal. Calcd for C20H25BN2O5: C, 62.52;
H, 6.56; N, 7.29. Found: C, 62.60; H, 6.59; N, 6.96.
2347
dx.doi.org/10.1021/jm101499u |J. Med. Chem. 2011, 54, 2341–2350

Journal of Medicinal Chemistry
ARTICLE
Table 5. Parameters for Itk, c-Kit, and Flt-3 Kinase Activity Assays
assay
[kinase] (nM)d
target peptide
SAM68e
[peptide]d
[ATP] (mM)d
μCi [γ-33P]ATP per nmol of ATP
time course (min)
Itka
c-Kitb
Flt-3c
25
30
1
3 μM
0.08
1.4
200
4
40
45
20
poly Glu/Tyr (4:1)
0.5 mg/mL
poly Glu/Tyr (4:1)
0.5 mg/mL
0.09
25
a Assay buffer: 20 mM MOPS (pH 7), 10 mM MgCl2, 1 mM DTT, 0.1% BSA. b Assay buffer: 100 mM HEPES (pH 7.5), 10 mM MgCl2, 25 mM NaCl, 1
mM DTT, 0.01% BSA. c Assay buffer: 100 mM HEPES (pH 7.5), 10 mM MgCl2, 25 mM NaCl, 1 mM DTT, 0.01% BSA. d Final assay concentration.
e Src-associated in mitosis 68 kDa protein, residues 332-443.
General Procedure for the Formation of Suzuki Adducts:
3-Benzyloxycarbonylamino-5-(2-(methylamino)pyrimidin-4-yl)-
(1H)-pyridin-2-one (5). 3-Benzyloxycarbonylamino-5-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)-(1H)-pyridin-2-one 4 (357 mg, 0.929 mmol,
1.0 equiv) was dissolved in toluene (15 mL) and ethanol (3 mL). 4-Chloro-
N-methylpyrimidin-2-amine (160 mg, 1.11 mmol, 1.2 equiv) was added, fol-
lowed by Pd(PPh3)4 (54 mg, 0.046 mmol, 5 mol %), sodium carbonate
(679 mg, 6.41 mmol, 6.9 equiv), andwater (6mL). The mixture washeated to
reflux for 2 h. After cooling, the reaction mixture was diluted with water and
extracted three times with ethyl acetate. The combined organics were washed
with brine, dried (MgSO4), and filtered, and the solvent was removed under
reduced pressure. The crude product was purified by flash chromatography on
silica, eluting with 40-70% ethyl acetate/petroleum ether to give 5 as a yellow
solid (227 mg, 0.62 mmol, 67%): 1HNMR(DMSO-d6) δ2.82(3H,s),3.94(3H,
s),5.17(2H,s),7.06(1H,d),7.11-7.19(1H,m),7.29-7.35 (5H, m), 8.30 (1H,
s), 8.58 (1H, s), 8.69 (1H, s), 9.10 (1H, s). MS m/z: 366.8 (M þ H)þ.
General Procedure for Cbz Deprotection: 3-Amino-5-
(2-(methylamino)pyrimidin-4-yl)-(1H)-pyridin-2-one (6). Asolu-
tion of 3-benzyloxycarbonylamino-5-(2-(methylamino)pyrimidin-4-
yl)-(1H)-pyridin-2-one 5 (200 mg, 0.548 mmol, 1.0 equiv) in ethyl
acetate (30 mL) and methanol (30 mL) was degassed under nitrogen,
and 10% Pd/C (40 mg, 20 wt %) was added. The mixture was stirred
under an atmosphere of hydrogen for 16 h before being filtered through
a pad of Celite, which was washed with methanol. The filtrate was
concentrated under reduced pressure to give 6 as a viscous oil (125 mg,
quantitative yield): 1H NMR (DMSO-d6) δ 2.86 (3H, d), 3.92 (3H, s),
5.15 (2H, br s), 6.98 (1H, d), 7.03 (1H, d), 7.56 (1H, br s), 8.09 (1H, s),
8.26 (1H, s). MS m/z: 232.7 (M þ H)þ.
3-Cyano-5-(pyridin-4-yl)-(1H)-pyridin-2-one (9). To a stirred
solution of 2-(pyridin-4-yl)malondialdehyde 8 (Acros Organics, 20 g,
134.2 mmol, 1.0 equiv) in benzene (500 mL) was added at ambient
temperature 2-cyanoacetamide (12.41 g, 147.5 mmol, 1.1 equiv), mor-
pholine (14.73 mL, 169 mmol, 1.26 equiv), and acetic acid (16 mL, 281.3
mmol, 2.1 equiv). The reaction mixture was heated to reflux for 24 h with
azeotropic removal of water using a Dean-Stark water separator. The
reaction mixture was cooled to ambient temperature and stirred for an
additional 24 h. The precipitated solid was isolated by filtration, washed
with ethyl acetate, and dried in vacuo. The crude product was triturated
from acetonitrile and the resultant precipitate isolated by filtration to give
1
9 as a brown solid (22.0 g, 111.7 mmol, 83%). H NMR (DMSO-d6)
δ 7.60 (2H, d), 8.26 (1H, s), 8.52 (2H, d), 8.59 (1H, s), 12.2 (1H, br s).
MS m/z: 198.2 (M þ H)þ.
3-Carboxy-5-(pyridin-4-yl)-(1H)-pyridin-2-one (10). A solu-
tion of 3-cyano-5-(pyridin-4-yl)-(1H)-pyridin-2-one 9 (22.0 g, 111.7
mmol, 1.0 equiv) in 9 N aqueous HCl (750 mL) was heated at reflux for
18 h. The reaction mixture was concentrated in vacuo and the residue
triturated from acetone. The resultant precipitate was isolated by filtration,
washed with acetone followed by ethanol, and dried in vacuo to give 10 as a
yellow solid (13 g, 60.1 mmol, 54%). 1H NMR (DMSO-d6) δ8.21 (2H, d),
8.64 (1H, s), 8.77 (2H, d), 8.82 (1H, d), 14.0 (1H, br s). MS m/z: 217.2
(M þ H)þ.
3-(4-(Piperidin-1-yl)phenylcarbamoyl)-5-(pyridin-4-yl)-
(1H)-pyridin-2-one (11). To a solution of 3-carboxy-5-(pyridin-4-
yl)-(1H)-pyridin-2-one 10 (303 mg, 1.4 mmol, 1.0 equiv) in dry DMF
(5 mL) were added at ambient temperature HOBT (236 mg, 1.54 mmol,
1.1 equiv) and TEA (234 μL, 1.68 mmol, 1.2 equiv) followed by EDCI
(295 mg, 1.54 mmol, 1.1 equiv). After the mixture was stirred for 15 min,
4-(piperidin-1-yl)aniline (296 mg, 1.68 mmol, 1.2 equiv) was added and
the reaction mixture was heated at 50 °C for 15 h. The reaction mixture
was diluted with water and the precipitated solid isolated by filtration
and washed successively with water, acetone, and diethyl ether. After
drying in vacuo, compound 11 was isolated as a yellow solid (105 mg,
0.28 mmol, 20%). 1H NMR (DMSO-d6) δ 1.48-1.68 (6H, m), 3.05-3.15
(4H, m), 6.93 (2H, d), 7.56 (2H, d), 7.72 (2H, d), 8.36 (1H, d), 8.61 (2H,
d), 8.80 (1H, d), 11.90 (1H, s), 13.20 (1H, brs). MSm/z: 375.5 (M þ H)þ.
HRMS calcd for C22H23N4O2, 375.1823; found, 375.1834.
Kinase Inhibition Assays. Materials and Reagents. HEPES,
MOPS, NaCl, MgCl2, BSA, ATP, TCA, NADH, poly Glu/Tyr, and
DMSO were supplied by Sigma-Aldrich. DTT was from Melford Labora-
tories. Phosphenol pyruvate, pyruvate kinase, and lactate dehydrogenase
were purchased from Roche diagnostics. Stock 3 mCi/mmol [γ-33P]ATP
and Optiphase Supermix scintillation cocktail were supplied by Perkin-
Elmer. Unifilter GF/C filter plates were from PerkinElmer.
General Procedure for the Preparation of Final Compounds
7a-y: 3-(4-(Piperidin-1-yl)benzoylamino)-5-(2-(methylamino)
pyrimidin-4-yl)-(1H)-pyridin-2-one (7o). To a solution of 4-
(piperidin-1-yl)benzoic acid (129 mg, 0.628 mmol, 1.0 equiv) in acetonitrile
(15 mL) was added 3-amino-5-(2-(methylamino)pyrimidin-4-yl)-(1H)-
pyridin-2-one 6 (145 mg, 0.628 mmol, 1.0 equiv) followed by phosphoryl
chloride (117 μL, 1.256 mmol, 2.0 equiv), and the mixture was heated to
reflux for 6 h. After the mixture was cooled, the solvent was removed under
reduced pressure and 2 M NaOH was added to the residue, followed by the
addition of 2 M HCl. The precipitate that formed was collected by filtra-
tion, washed with water, and then recrystallized from methanol to give
3-(4-(piperidin-1-yl)benzoylamino)-5-(2-(methylamino)pyrimidin-4-yl)-
(1H)-pyridin-2-one 7o (163 mg, 0.40 mmol, 55%).
(S)-3-(4-(2-(Pyrrolidin-1-ylmethyl)pyrrolidin-1-yl)benzoyla-
mino)-5-(6-(methylamino)pyrazin-2-yl)-(1H)-pyridin-2-one
1
(7v). H NMR (DMSO-d6) δ 1.69 (br s, 4H), 1.90-2.08 (m, 4H),
2.37-40 (m, 1H), 2.40-2.45 (m, 1H), 2.59 (br d, 2H), 2.87 (d, 3H),
2.96 (br s, 2H), 3.12-3.19 (m, 1H), 3.41-3.45 (m, 1H), 3.93 (br s, 1H),
6.66 (d, 2H), 7.08-7.11 (m, 1H), 7.75-7.79 (m, 3H), 7.84-7.86
(m, 1H), 8.08 (s, 1H), 8.98 (d, 1H), 9.04 (s, 1H), 12.42 (d, 1H). MS
m/z: 474.4 (M þ H)þ. HRMS calcd for C26H32N7O2, 474.2619; found,
474.2612.
Itk, c-Kit, and Flt-3 Kinase Inhibition Assays. Ability of compounds to
inhibit Itk, c-Kit, or Flt-3 kinase activity was tested using a radiometric
phosphate incorporation assay as described in Table 5 and below.
A stock solution was prepared consisting of the appropriate buffer, kinase,
and target peptide. To this was added the compound of interest at varying
concentrations in DMSO to a final DMSO concentration of 3%. Assays were
initiated by addition of an appropriate [γ-33P]ATP solution and incubated at
25 °C. Assays were stopped, after the desired time course, by addition of TCA
(1H)-Pyridin-2-ones 7a-y. Analytical data are provided in the
Supporting Information.
2348
dx.doi.org/10.1021/jm101499u |J. Med. Chem. 2011, 54, 2341–2350

Journal of Medicinal Chemistry
ARTICLE
and ATP to a final concentration of 10% and 0.2 mM, respectively. Peptides
were captured on a Unifilter GF/C filter 96-well plate prepared according to
the manufacturer’s instructions and washed three times with 200 μL of 5%
TCA prior to addition of 50 μL of scintillation cocktail and scintillation
counting on a 1450 Microbeta liquid scintillation counter (Perkin Elmer).
Dose-response data were analyzed using GraphPad Prism software (version
3.0cx for Macintosh, GraphPad software).
(3) Fowell, D. J.; Shinkai, K.; Liao, X. C.; Beebe, A. M.; Coffman,
R. L.; Littman, D. R.; Locksley, R. M. Impaired NFATc translocation and
failure of Th2 development in Itk-deficient CD4þ T cells. Immunity
1999, 11, 399–409.
(4) Schaeffer, E. M.; Debnath, J.; Yap, G.; McVicar, D.; Liao, X. C.;
Littman, D. R.; Sher, A.; Varmus, H. E.; Lenardo, M. J.; Schwartzberg,
P. L. Requirement for Tec kinases Rlk and Itk in T cell receptor signaling
and immunity. Science 1999, 284, 638–641.
Src Kinase Inhibition Assay. Reactions were carried out at 30 °C in a total
volume of 67 μL in a reaction buffer containing 25 mM HEPES (pH 7.5), 20
mM MgCl2, 2 mM DTT, 280 μM NADH, 1 mM phosphoenol pyruvate, 30
μg/mL pyruvate kinase, 10 μg/mL lactate dehydrogenase, 3% DMSO, 50
μM ATP, 0.3 mg/mL poly Glu/Tyr (4:1), 25 nM Src, and serially diluted
inhibitor compounds. Initial rate data were determined from the rate of
change of absorbance at 340 nM (corresponding to stoichiometric con-
sumption of NADH) using a Molecular Devices Spectramax plate reader
(Sunnyvale, CA) over 10 min at 30 °C. Dose-response data were analyzed
using GraphPad Prism software (version 3.0cx for Macintosh, GraphPad
software).
(5) Schaeffer, E. M.; Yap, G. S.; Lewis, C. M.; Czar, M. J.; McVicar,
D. W.; Cheever, A. W.; Sher, A.; Schwartzberg, P. L. Mutation of Tec
family kinases alters T helper cell differentiation. Nat. Immunol. 2001,
2, 1183–1188.
(6) Mueller, C.; August, A. Attenuation of immunological symptoms
of allergic asthma in mice lacking the tyrosine kinase ITK. J. Immunol.
2003, 170, 5056–5063.
(7) Matsumoto, Y.; Oshida, T.; Obayashi, I.; Imai, Y.; Matsui, K.;
Yoshida, N. L.; Nagata, N.; Ogawa, K.; Obayashi, M.; Kashiwabara, T.;
Gunji, S.; Nagasu, T.; Sugita, Y.; Tanaka, T.; Tsujimoto, G.; Katsunuma,
T.; Akasawa, A.; Saito, H. Identification of highly expressed genes in
peripheral blood T cells from patients with atopic dermatitis. Int. Arch.
Allergy Immunol. 2002, 129, 327–340.
(8) Sahu, N.; August, A. ITK inhibitors in inflammation and
immune-mediated disorders. Curr. Top. Med. Chem. 2009, 9, 690–703.
(9) Lo, H.-Y. Itk inhibitors: a patent review. Expert Opin. Ther. Pat.
2010, 20, 459–469.
(10) Das, J.; Chunjian, L.; Moquin, R. V.; Lin, J.; Furch, J. A.; Spergel,
S. H.; McIntyre, K. W.; Shuster, D. J.; O’Day, K. D.; Penhallow, B.;
Hung, C.-Y.; Kanner, S. B.; Lin, T.-A.; Dodd, J. H.; Barrish, J. C.; Wityak,
J. Discovery and SAR of 2-amino-5-[(thiomethyl)aryl]thiazoles as
potent and selective Itk inhibitors. Bioorg. Med. Chem. Lett. 2006,
16, 2411–2415.
(11) Das, J.; Furch, J. A.; Liu, C.; Moquin, R. V.; Lin, J.; Spergel,
S. H.; McIntyre, K. W.; Shuster, D. J.; O’Day, K. D.; Penhallow, B.;
Hung, C.-Y.; Doweyko, A. M.; Kamath, A.; Zhang, H.; Marathe, P.;
Kanner, S. B.; Lin, T.-A.; Dodd, J. H.; Barrish, J. C.; Wityak, J. Discovery
and SAR of 2-amino-5-(thioaryl)thiazoles as potent and selective Itk
inhibitors. Biorg. Med. Chem. Lett. 2006, 16, 3706–3712.
(12) Snow, R. J.; Abeywardane, A.; Campbell, S.; Lord, J.; Kashem,
M. A.; Khine, H. H.; King, J.; Kowalski, J. A.; Pullen, S. S.; Roma, T.;
Roth, G. P.; Sarko, C. R.; Wilson, N. S.; Winters, M. P.; Wolak, J. P.;
Cywin, C. L. Hit-to-lead studies on benzimidazole inhibitors of ITK:
discovery of a novel class of kinase inhibitors. Biorg. Med. Chem. Lett.
2007, 17, 3660–3665.
(13) Moriarty, K. J.; Winters, M.; Qiao, L.; Ryan, D.; DesJarlis, R.;
Robinson, D.; Cook, B. N.; Kashem, M. A.; Kaplita, P. V.; Liu, L. H.;
Farrell, T. M.; Khine, H. H.; King, J.; Pullen, S. S.; Roth, G. P.; Magolda,
R.; Takahashi, H. Itk kinase inhibitors: initial efforts to improve the
metabolical stability and the cell activity of the benzimidazole lead.
Bioorg. Med. Chem. Lett. 2008, 18, 5537–5540.
’ ASSOCIATED CONTENT
S
Supporting Information. Analytical data for 7a-y, sum-
b
mary of biological evaluation for 7a-y and 11, and details of the
assays used. This material is available free of charge via the Internet
at http://pubs.acs.org.
Accession Codes
†Coordinates of 7a and 7o cocrystallized with Itk have been
submitted to the RCSB Protein Data Bank (PDB codes 3QGW
and 3QGY, respectively).
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ 44 (0) 1235 438800. Fax: þ 44 (0) 1235 820440.
E-mail: jean-damien_charrier@vrtx.com.
’ ACKNOWLEDGMENT
We thank C. Hudson, C. Memmott, C. Stuver-Moody,
P. Wang, and J. Westcott for support with biochemical studies,
G. Cheetham for structural information, the entire Itk project
team, and S. Young for valuable discussions.
’ ABBREVIATIONS USED
ATP, adenosine 50-triphosphate; Bmx, bone marrow tyrosine
kinase gene in chromosome X protein; Btk, Bruton’s tyrosine
kinase; BI, Boehringer Ingelheim;BMS, Bristol-Myers Squibb;Cbz,
carbobenzyloxy; CDK2, cyclin-dependent kinase 2; Flt3, FMS-like
tyrosine kinase 3; HTS, high throughput screening; IFN-γ, interfer-
on-γ; IL-2, interleukin-2; IL-4, interleukin-4; IL-5, interleukin-5; IL-
10, interleukin-10; Itk, interleukin-2 inducible T-cell kinase; LE,
ligand efficiency; PLC-γ, phospholipase C γ; Rlk, resting lympho
cyte kinase; Tec, tyrosine kinase expressed in hepatocellular carcino-
ma; TCR, T-cell receptor; SAR, structure-activity relationship
(14) Moriarty, K. J.; Takahashi, H.; Pullen, S. S.; Khine, H. H.; Sallati,
R. H.; Raymond, E. L.; Woska, J. R., Jr.; Jeanfavre, D. D.; Roth, G. P.;
Winters, M. P.; Qiao, L.; Ryan, D.; DesJarlais, R.; Robinson, D.; Wilson,
M.; Bobko, M.; Cook, B. N.; Yin, Lo, H.; Nemoto, P. A.; Kashem, M. A.;
Wolak, J. P.; White, A.; Magolda, R. L.; Tomczuk, B. Discovery, SAR and
X-ray structure of 1H-benzimidazole-5-carboxylic acid cyclohexyl-methyl-
amides as inhibitors of inducible T-cell kinase (Itk). Bioorg. Med. Chem. Lett.
2008, 18, 5545–5549.
(15) Cook, B. N.; Bentzien, J.; White, A.; Nemoto, P. A.; Wang, J.;
Man, C. C.; Soleymanzadeh, F.; Khine, H. H.; Kashem, M. A.; Kugler,
S. Z., Jr.; Wolak, J. P.; Roth, G. P.; De Lombaert, S.; Pullen, S. S.;
Takahashi, H. Discovery of potent inhibitors of interleukin-2 inducible
T-cell kinase (ITK) through structure-based drug design. Bioorg. Med.
Chem. Lett. 2009, 19, 773–777.
’ REFERENCES
(1) Berg, L. J.; Finkelstein, L. D.; Lucas, J. A.; Schwartzberg, P. L. Tec
family kinases in T lymphocyte development and function. Annu. Rev.
Immunol. 2005, 23, 549–600.
(2) Liao, X. C.; Littman, D. R. Altered T cell receptor signaling and
disrupted T cell development in mice lacking Itk. Immunity 1995,
3, 757–769.
(16) Winters, M. P.; Robinson, D. J.; Khine, H. H.; Pullen, S. S.;
Woska, J. R., Jr.; Raymond, E. L.; Sellati, R.; Cywin, C. L.; Snow, R. J.;
Kashem, M. A.; Wolak, J. P.; King, J.; Kaplita, P. V.; Liu, L. H.; Farrell,
T. M.; DesJarlais, R.; Roth, G. P.; Takahashi, H.; Moriarty, K. J.
5-Aminomethyl-1H-benzimidazoles as orally active inhibitors of
2349
dx.doi.org/10.1021/jm101499u |J. Med. Chem. 2011, 54, 2341–2350

Journal of Medicinal Chemistry
ARTICLE
inducible T-cell kinase (Itk). Bioorg. Med. Chem. Lett. 2008,
18, 5541–5544.
(17) Riether, D.; Zindell, R.; Kowalski, J. A.; Cook, B. N.; Bentzien, J.;
De Lombaert, S.; Thomson, D.; Kugler, S. Z., Jr.; Skow, D.; Martin, L. S.;
Raymond, E. L.; Khine, H. H.; O’Shea, K.; Woska, J. R., Jr.; Jeanfavre, D.;
Sellati, R.; Ralph, K. L. M.; Ahlberg, J.; Labissiere, G.; Kashem, M. A.;
Pullen, S. S.; Takahashi, H. 5-Aminomethylbenzimidazoles as potent
ITK antagonists. Bioorg. Med. Chem. Lett. 2009, 19, 1588–1591.
(18) Lin, T. A.; McIntyre, K. W.; Das, J.; Liu, C.; O’Day, K. D.;
Penhallow, B.; Hung, C. Y.; Whitney, G. S.; Shuster, D. J.; Yang, X.;
Townsend, R.; Postelnek, J.; Spergel, S. H.; Lin, J.; Moquin, R. V.; Furch,
J. A.; Kamath, A. V.; Zhang, H.; Marathe, P. H.; Perez-Villar, J. J.;
Doweyko, A.; Killar, L.; Dodd, J. H.; Barrish, J. C.; Wityak, J.; Kanner,
S. B. Selective Itk inhibitors block T-cell activation and murine lung
inflammation. Biochemistry 2004, 43, 11056–11062.
(19) Li, R.; Xue, L.; Zhu, T.; Jiang, Q.; Cui, X.; Yan, Z.; McGee, D.;
Wang, J.; Gantla, V. R.; Pickens, J. C.; McGrath, D.; Chucholowski, A.;
Morris, S. W.; Webb, T. R. Design and synthesis of 5-aryl-pyridone-
carboxamides as inhibitors of anaplastic lymphoma kinase. J. Med. Chem.
2006, 49, 1006–1015.
(20) Kuethe, J.; Wong, A.; Davis, I. W. Rapid and efficient synthesis
of 1H-indol-2-yl-1H-quinolin-2-ones. Org. Lett. 2003, 5, 3975–3978.
(21) Kiethe, J. Y.; Wong, A.; Qu, C.; Smitrovitch, J.; Davies, I. W.;
Hughes, D. L. Synthesis of 5-substituted-1H-indol-2-yl-1H-quinolin-2-
ones: a novel class of KDR kinase inhibitors. J. Org. Chem. 2005,
70, 2555–2567.
(22) Durrant, S. J.; Pinder, J. L.; Charrier, J.- D.; Jimenez, J.- M.;
Brenchey, G.; Collier, P. N.; Kay, D.; Miller, A.; Pierard, F.; Ramaya, S.;
Sadiq, S.; Twin, H. Synthesis of a stable pyridyl boronate and its reaction
with aryl and heteroaryl halides. Heterocycles 2006, 70, 509–517.
(23) Zhu, L.; Duquette, J.; Zhang, M. An improved preparation of
arylboronates: application in one-pot Suzuki biaryl synthesis. J. Org.
Chem. 2003, 68, 3729–3732.
(24) Brown, K.; Long, J. M.; Vial, S. C.; Dedi, N.; Dunster, N. J.;
Renwick, S. B.; Tanner, A. J.; Frantz, J. D.; Fleming, M. A.; Cheetham,
G. M. Crystal structures of interleukin-2 tyrosine kinase and their
implications for the design of selective inhibitors. J. Biol. Chem. 2004,
279, 18727–32.
(25) Gajiwala, K. S.; Wu, J. C.; Christensen, J.; Deshmukh, G. D.;
Diehl, W.; DiNitto, J. P.; English, J. M.; Greig, M. J.; He, Y. A.; Jacques,
S. L.; Lunney, E. A.; McTigue, M.; Molina, D.; Quenzer, T.; Wells, P. A.;
Yu, X.; Zhang, Y.; Zou, A.; Emmett, M. R.; Marshall, A. G.; Zhang, H. M.;
Demetri, G. D. KIT kinase mutants show unique mechanisms of drug
resistance to imatinib and sunitinib in gastrointestinal stromal tumor
patients. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 1542–1547.
(26) Mol, C. D.; Dougan, D. R.; Schneider, T. R.; Skene, R. J.; Kraus,
M. L.; Scheibe, D. N.; Snell, G. P.; Zou, H.; Sang, B. C.; Wilson, K. P.
Structural basis for the autoinhibition and STI-571 inhibition of c-Kit
tyrosine kinase. J. Biol. Chem. 2004, 279, 31655–31663.
(27) Mol, C. D.; Lim, K. B.; Sridhar, V.; Zou, H.; Chien, E. Y.; Sang,
B. C.; Nowakowski, J.; Kassel, D. B.; Cronin, C. N.; McRee, D. E.
Structure of a c-kit product complex reveals the basis for kinase
transactivation. J. Biol. Chem. 2003, 278, 31461–31464.
(28) Griffith, J.; Black, J.; Faerman, C.; Swenson, L.; Wynn, M.; Lu,
F.; Lippke, J.; Saxena, K. The structural basis for autoinhibition of FLT3
by the juxtamembrane domain. Mol. Cell 2004, 13, 169–178.
(29) Abel, R.; Young, T.; Farid, R.; Berne, B. J.; Friesner, R. A. Role
of the active-site solvent in the thermodynamics of factor Xa ligand
binding. J. Am. Chem. Soc. 2008, 130, 2817–2831.
(30) Knegtel, R. M. A.; Robinson, D. Unpublished results.
(31) Howard, J. A. K.; Hoy, V. J.; O’Hagan, D.; Smith, G. T. How
good is fluorine as a hydrogen bond acceptor?. Tetrahedron 1996,
52, 12613–12622.
(32) Manning, G.; Whyte, D. B.; Martinez, R; Hunter, T.; Sudarsanam,
S. The protein kinase complement of the human genome. Science 2002,
298, 1912–1934.
2350
dx.doi.org/10.1021/jm101499u |J. Med. Chem. 2011, 54, 2341–2350