Discovery of aryl aminoquinazoline pyridones as potent, selective, and orally efficacious inhibitors of receptor tyrosine kinase c-Kit

Article abstract of DOI:10.1021/jm800188g

Inhibition of c-Kit has the potential to treat mast cell associated fibrotic diseases. We report the discovery of several aminoquinazoline pyridones that are potent inhibitors of c-Kit with greater than 200-fold selectivity against KDR, p38, Lck, and Src.

Full text of DOI:10.1021/jm800188g

J. Med. Chem. 2008, 51, 3065–3068
3065
Table 1. Comparison of Kinase Selectivity
Discovery of Aryl Aminoquinazoline Pyridones as
Potent, Selective, and Orally Efficacious
Inhibitors of Receptor Tyrosine Kinase c-Kit†
Essa Hu,*,‡ Andrew Tasker,‡ Ryan D. White,‡
Roxanne K. Kunz,‡ Jason Human,‡ Ning Chen,‡
Roland Bürli,‡ Randall Hungate,‡ Perry Novak,§
Andrea Itano,| Xuxia Zhang,| Violeta Yu, Yen Nguyen,
Yanyan Tudor, Matthew Plant,| Shaun Flynn, Yang Xu,#
Kristin L. Meagher,3 Douglas A. Whittington,3 and
Gordon Y. Ng|
kinase IC50, (µM)a
1
2
10
c-Kit
KDR
p38
Lck
Src
0.0061
0.048
0.20
0.019
0.40
0.077
>25
0.99
1.9
0.0010
1.4
5.8
0.58
0.80
Department of Medicinal Chemistry, Department of Inflammation,
Department of HTS and Molecular Pharmacology, Department of
Pharmacokinetics and Drug Metabolism, Department of Molecular
Structures, Amgen Inc., One Amgen Center DriVe, Thousand Oaks,
California 91320-1799 Department of Medicinal Chemistry,
Department of Small Molecule Process DeVelopment, Amgen Inc.,
One Kendall Square, Building 1000,
5.0
a Potency data is reported as an average of 2 runs.
Essential for mast cell survival, proliferation, activation, and
chemotaxis is the activation of c-Kit receptors expressed on the
surface of mast cells.6 c-Kit is a member of the type III receptor
tyrosine kinase family along with colony-stimulating factor-1
(cFMS) and platelet-derived growth factor receptor (PDGFR).
c-Kit is activated upon binding of its cognate ligand, stem cell
factor. Excessive levels of SCF or autoactivation of c-Kit, due
to gain-of-function mutations, have been linked to mastocytosis.6
The association between c-Kit, mast cells, and fibrosis is
supported by several preclinical studies conducted with imatinib
mesylate, a multikinase inhibitor that inhibited c-Kit.7 In the
clinic, imatinib treatment reduced bone marrow fibrosis in
patients with chronic myelogenous leukemia.8 Clinical evalu-
ation of imatinib in IPF patients is currently underway.
We were interested in developing a potent and selective c-Kit
inhibitor for the treatment of fibrotic diseases. To enhance the
safety margin, we decided that a more selective c-Kit inhibitor
that does not interfere with kinases associated with angiogenesis
and immune cell signaling pathways, such as KDR, p38, Lck,
and Src, was highly desired.
Our investigation began with novel pyridone 1, which was
identified by a screen of our proprietary kinase preferred library.
Pyridone 1 was a nonselective inhibitor of c-Kit (IC50 ) 6.1
nM, Table 1). This report describes the structure-activity
relationship investigations that led to the discovery of potent
c-Kit inhibitors that exhibited greater than 200-fold kinase
selectivity against KDR, p38, Lck, and Src. Inhibition of c-Kit
phosphorylation was measured by a homogeneous time-resolved
fluorescent kinase assay and a SCF stimulated UT7 cell
proliferation/survival assay. In vivo efficacy was assessed in a
rodent pharmacodynamic model of mast cell activation to
demonstrate proof-of-concept.
Cambridge, Massachusetts 02139
ReceiVed February 21, 2008
Abstract: Inhibition of c-Kit has the potential to treat mast cell
associated fibrotic diseases. We report the discovery of several
aminoquinazoline pyridones that are potent inhibitors of c-Kit with
greater than 200-fold selectivity against KDR, p38, Lck, and Src. In
vivo efficacy of pyridone 16 by dose-dependent inhibition of histamine
release was demonstrated in a rodent pharmacodynamic model of mast
cell activation.
Mast cells have been implicated in several fibrotic diseases
such as scleroderma and idiopathic pulmonary fibrosis (IPFa).1
These are grievous diseases with high mortality rates. Patients
with IPF, for example, have a mean life span of 3-8 years from
onset of disease. Mast cells are predominantly expressed beneath
epithelia surfaces, in the mucosal and connective tissues of the
skin, the airways, and the intestines. Considered as effector cells
in allergy, inflammation, autoimmunity, and fibrosis, these cells
are a major source of preformed TNF, pro-inflammatory, and
profibrotic mediators.2 In fibrosis, activated mast cells release
mediators to promote fibroblast proliferation.1 Reciprocally,
fibroblasts secrete stem cell factor (SCF), also known as mast
cell growth factor or Steel ligand, to induce further mast cell
expansion and activation.3 Excessive elevation of mast cell
burden has been linked to various fibrotic diseases.4 Further-
more, attenuation of fibrosis has been reported in mast cell
deficient mice.5 Therefore, regulation of mast cell proliferation
and function may have therapeutic potential in fibrotic diseases.
The general synthetic strategy for these pyridone analogues
is illustrated in Scheme 1. In a two-step, one-pot protocol
developed by Hynes and Campbell, 5-bromo-2-fluorobenzal-
dehyde 3 was reacted with guanidine carbonate at 160 °C to
form 2-amino-6-bromoquinazoline 4.9 Miyaura conditions con-
verted the aryl bromide to boronic ester 5.10 Preparation of PMB
protected pyridine 7 was accomplished by reacting 3-bromo-
2-chloro-4-methylpyridine 6 with (4-methoxyphenyl)methanol.
Suzuki coupling of aminoquinazoline boronic ester 5 with bromo
pyridine 7 afforded aminoquinazoline pyridine 8. Deprotection
with trifluoroacetic acid produced pyridone 9. Selective N-
alkylation was achieved with sodium hydride to form alkyl
pyridone 10. Buchwald’s copper-catalyzed aryl amidation
provided aryl pyridones 11-19 in good yield.11
†
Atomic coordinates and structure factors for cocrystal structures of
compounds 1, 2, and 16 with V916T KDR can be accessed using PDB
codes 3CP9, 3CPB, and 3CPC, respectively.
* To whom correspondence should be addressed. Phone: 805-313-5300.
Fax: 805-480-3016. E-mail: ehu@amgen.com. Address: Amgen Inc., One
Amgen Center Drive, Thousand Oaks, California 91320-1799.
‡
Department of Medicinal Chemistry, Amgen Inc.
Department of Small Molecule Process Development, Amgen Inc.
Department of Inflammation, Amgen Inc.
Department of HTS and Molecular Phamacology, Amgen Inc.
Department of Pharmacokinetics and Drug Metabolism, Amgen Inc.
Department of Molecular Structures, Amgen Inc.
§
|
#
3
a Abbreviations: IPF, idiopathic pulmonary fibrosis; cFMS, colony-
stimulating factor; PDGFR, platelet-derived growth factor receptor; SCF,
stem cell factor; PMB, para-methoxy benzyl; SAR, structure-activity
relationship; PK, pharmacokinetic; CL, clearance.
10.1021/jm800188g CCC: $40.75
2008 American Chemical Society
Published on Web 05/01/2008

3066 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Scheme 1. General Preparation of Aryl Pyridonesa
Letters
selectivity was not evident based on the co-crystal structure.
Nevertheless, overlay of co-crystal structures of 1 and 2 in the
V916T mutant KDR protein (a close analog of c-Kit) showed
that the aliphatic section of 2 was also the region where the
molecules most differed (Figure 1). We hypothesized that
attaching a similar alkyl group to the pyridone scaffold could
improve its kinase selectivity. Indeed, alkyl pyridones such as
cyclopentylethyl 10 did obtain greater than 500-fold kinase
selectivity. However, these pyridones also exhibited high in vivo
clearance in rats. Attempts to enhance metabolic stability by
adding heteroatoms to the alkyl groups decreased c-Kit activity.
Based on the overlapped structures, we then speculated that
an investigation of substitutions on the 3- and 4- positions of
the aryl pyridones could improve kinase selectivity. Furthermore,
pharmacokinetic profiles of these pyridones might be more
desirable. Thus, a systematic examination of the aryl pyridones
was undertaken (Table 2). Devoid of any substitution on the
lower aromatic ring, 11 exhibited decreased c-Kit activity (IC50
) 0.21 µM). Mimicking the gem-dimethyl group on 1 with a
tert-butyl group at the 4-position, 12 regained c-Kit potency
(IC50 ) 2.1 nM) but exhibited poor selectivity. Further decreas-
ing the steric bulk from trifluoromethyl group (13) to methoxy
group (14) or chlorine (15) notably reduced inhibitory activity
against KDR and p38 to the micromolar range. Regioisomer
16 exhibited comparable c-Kit potency and kinase selectivity.
However, a nearly 4-fold decrease in c-Kit activity was observed
with 17. Whereas 3-fluoro-4-chloro substitution (18) reduced
c-Kit potency, 3-fluoro-4-methyl substitution (19) afforded a
potent c-Kit inhibitor with at least 125-fold selectivity and
greater than 5 µM activity against KDR, p38, Lck, and Src.13
Switching the positions of the methyl and fluoro groups (20)
decreased kinase selectivity against Lck and Src.
a Reaction conditions: (a) guanidine carbonate, DIPEA, NMP, 160°C,
(48%); (b) bis(pinacolato)diboron, KOAc, PdCl2(dppf)·CH2Cl2, 85°C,
(50%); (c) NaH, PMBOH, (96%); (d) Pd(OAc)2, S-phos, K3PO4 (90%);
(e) TFA; (f) NaH, 2-cyclopentylethyl 4-methylbenzenesulfonate, 70°C
(20%); (g) CuI, K3PO4, DMEDA, ArI, NMP, 85°C (60-80% two steps).
Unfortunately, pyridone 19 also exhibited high clearance in
rats (CL ) 1.95 L/h/kg). With its 4-substituted methyl group a
likely metabolic liability by oxidation, we examined alternative
substituents at the 4-position to improve clearance. A repre-
sentative subset of the analogs examined is shown in Table 3.
Acetyl (21) and ethyl ester (22) replacements decreased
selectivity against Lck and Src. While morpholine (23) restored
Lck and Src activity to the micromolar range, N-linked pyrazole
(24) reduced selectivity against Lck and Src. Fortunately,
oxazole 25 resulted in both excellent c-Kit potency (enzyme
IC50 ) 22 nM, cell IC50 ) 16 nM) and greater than 200-fold
Figure 1. Overlap of co-crystal structures of pyridone 1 and bisamide
2 in V916T KDR mutant protein.
Our first approach to improve the kinase selectivity was to
compare pyridone 1 to bisamide 2, a more selective c-Kit
inhibitor we recently reported (Table 1).12 The aliphatic group
on 2 was necessary for kinase selectivity, but the reason for the
Table 2. SAR of Phenyl Ring Substitutions
c-Kit Enzyme
c-Kit cell IC50
KDR IC50
p38 IC50
Lck IC50
Src IC50
cmpd
R2
R3
IC50(µM)a
(µM)a
(µM)a
(µM)a
(µM)a
(µM)a
11
12
13
14
15
16
17
18
19
20
H
H
H
H
H
0.21
>1.0
0.011
1.8
>5.0
>5.0
>5.0
>5.0
>5.0
>5.0
4.3
1.7
0.0072
1.2
0.15
0.34
1.7
0.41
0.48
25
0.99
0.036
0.75
0.15
0.26
0.86
0.27
0.39
25
tBu
CF3
OMe
Cl
0.0021
0.0079
0.017
0.020
0.014
0.065
0.060
0.040
0.014
0.016
0.018
0.018
0.014
0.028
0.028
0.022
0.012
0.018
0.028
3.9
6.5
4.9
6.1
8.0
9.2
40
4.5
H
CF3
OMe
F
F
Me
H
H
Cl
Me
F
0.74
0.043
a Potency data is reported as an average of 2 runs.

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3067
Table 3. SAR of 4-Substituted Pyridonesa
Figure 3. Pyridone 16 decreased SCF-induced release of histamine
in a dose-dependent manner.
Table 5. Comparison of Imatinib and Pyridones 16 and 25
a Potency data is reported as an average of 2 runs.
Table 4. Comparison of Rat Pharmacokinetic Profilesa
b
c
c
c
CLb
Vdss
AUC0-t
Cmax
T1/2
Fc
cmpd
(L/h/kg)
(L/kg)
(ng*h/mL)
(ng/mL)
(h)
(%)
16
19
25
2.46
1.95
0.46
4.12
2.39
1.59
1036
1309d
9860
253
280d
1230
1.83
2.9d
2.6
25
100d
39
a Male Sprague-Dawley rats (n ) 3). b iv, 1.0 mg/kg (DMSO). c po,
10.0 mg/kg (2% HPMC, 1% Tween 80, pH 2.0 with HCl). d po, 2.0 mg/kg
(2% HPMC, 1% Tween 80, pH 2.0 with HCl).
a Potency data is reported as an average of 2 runs. b BE ) binding
efficiency.
mast cells in vivo following systemic administration of recombinant
human stem cell factor has been reported in the clinic. Moreover,
SCF administration to nonhuman primate results in mast cell
hyperplasia.14 Thus, we used recombinant rodent SCF to promote
mast cell activation in mice and assessed the extent of activation
by measuring histamine release in the blood stream. The study
began with a single oral administration of pyridone 16 to female
C57BL/6 mice at 100, 20, and 5 mg/kg. One hour postdosing, 80
µg/kg of SCF was administered by intravenous injection. Fifteen
minutes after SCF treatment, the mice were euthanized and blood
was collected by cardiac puncture for histamine measurement and
PK analysis. As shown in Figure 3, a dose-dependent decrease in
SCF-induced histamine release was observed with ED50 of 4.7 mg/
kg. With mouse protein binding of 87.85% by ultracentrifugation,
free fraction EC50 was 12.6 nM, comparable to cell IC50 of 28 nM
for 16.15
Compared to imatinib, pyridones 16 and 25 exhibited compa-
rable or better potency against c-Kit with approximately two-thirds
of the molecular weight of imatinib (Table 5). Besides higher
binding efficiency,16 these two compounds also achieved improved
selectivity against off-target kinases such as p38 and Lck. Both
pyridones also exhibited better selectivity against Abl, while 16
showed comparable selectivity against PDGFR compared to
imatinib. Better selectivity against Abl may be desirable given a
reported link between cardiotoxicity and inhibition of Abl.17
Because the residues comprising the ATP binding pocket of c-Kit
and PDGFR are over 98% homologous, selectivity against PDGFR
will likely be difficult to achieve.18 To date, a c-Kit inhibitor with
significant PDGFR selectivity has not been reported. Meanwhile,
several studies have suggested that inhibition of PDGFR may be
beneficial for treatment of certain fibrotic conditions.19 Thus, a c-Kit
Figure 2. Co-crystal structure of 16 in mutant KDR (V916T), a close
analog of c-Kit. mKDR residue numbers in black, and c-Kit residue
numbers in blue.
selectivity against KDR, p38, Lck, and Src. Furthermore,
clearance in rats also improved (CL ) 0.46 L/h/kg, Table 4).
Although obtaining co-crystals of these pyridones in the c-Kit
protein has been challenging, we succeeded in crystallizing 16 in
mutant KDR (V916T). By changing the gatekeeper residue from
valine to threonine, this mutant shares the majority of residues with
c-Kit in the ATP binding pocket. On the basis of the co-crystal
structure, we were able to infer the key interactions the compound
would make in c-Kit (Figure 2). Occupying the ATP binding site,
the inhibitor binds in the “DFG-out” conformation. Two nitrogens
from the aminoquinazoline portion of the molecule form hydrogen
bonding interactions with linker region of the enzyme at Cys673.
The pyridone scaffold is further anchored by the hydrogen bond
contacts between its carbonyl group and Asp810.
To evaluate the efficacy of these c-Kit inhibitors in vivo,
pyridone 16 was tested in a clinically relevant rodent pharmaco-
dynamic model of mast cell activation. Functional activation of

3068 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Letters
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C.; Buchanan, J. L.; Buckner, W. H.; Cee, V. J.; Chai, L.; Deak, H. L.;
Epstein, L. F.; Faust, T.; Gallant, P.; Geuns-Meyer, S. D.; Gore, A.;
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inhibitor with PDGFR activity but lacking KDR, p38, Lck, and
Src activity may be desired. Further internal kinase screens showed
that 16 and 25 were also selective against 36 and 41 other kinases,
respectively.20
In conclusion, starting with a novel pyridone 1, our SAR
efforts resulted in the identification of potent, selective, and
orally efficacious c-Kit inhibitors. Improvements in kinase
selectivity were achieved with alkyl pyridone 10 and aryl
pyridones 16, 19, and 25. Pyridone 25 exhibited potent inhibition
of c-Kit, greater than 200-fold selectivity against KDR, p38,
Lck, and Src, and desirable pharmacokinetic properties. Oral
efficacy in vivo was demonstrated in a clinically relevant rodent
pharmacodynamic model of mast cell activation. Efficacy of
c-Kit inhibition in a wound fibrogenesis model of mast cell
activation and expansion will be reported in a separate publica-
tion.21 These findings suggest that the pyridones are promising
therapeutic compounds for the treatment of fibrotic diseases.
Supporting Information Available: Experimental details and
characterization of all compounds, biological methods as well as
X-ray crystal data for 1, 2, and 16. This material is available free
of charge via the Internet at http://pubs.acs.org.
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(19) (a) Hoyle, G. W.; Li, J.; Finkelstein, J. B.; Eisenberg, T.; Liu, J. Y.;
Lasky, J. A.; Athas, G.; Morris, G. F.; Brody, A. R. Emphysematous
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(20) All kinases were tested at their apparent Km of ATP with respect to
1 µM of peptide substrate at single point. Selectivity was defined as
exhibiting greater than 50% of control against target kinases. See
Supporting Information for list of kinases screened.
(21) Zoog, S. J.; Itano, A.; Trueblood, E.; Pacheco, E.; Ferbas, J.; Ng, G. Y.;
Juan, G. unpublished results.
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