Hybrid compound design to overcome the gatekeeper T338M mutation in cSrc

Article abstract of DOI:10.1021/jm9002928

The emergence of drug resistance remains a fundamental challenge in the development of kinase inhibitors that are effective over long-term treatments. Allosteric inhibitors that bind to sites lying outside the highly conserved ATP pocket are thought to be more selective than ATP-competitive inhibitors and may circumvent some mechanisms of drug resistance. Crystal structures of type I and allosteric type III inhibitors in complex with the tyrosine kinase cSrc allowed us to employ principles of structure-based design to develop these scaffolds into potent type II kinase inhibitors. One of these compounds, 3c (RL46), disrupts FAK-mediated focal adhesions in cancer cells via direct inhibition of cSrc. Details gleaned from crystal structures revealed a key feature of a subset of these compounds, a surprising flexibility in the vicinity of the gatekeeper residue that allows these compounds to overcome a dasatinib-resistant gatekeeper mutation emerging in cSrc.

Full text of DOI:10.1021/jm9002928

J. Med. Chem. 2009, 52, 3915–3926
3915
Hybrid Compound Design To Overcome the Gatekeeper T338M Mutation in cSrc#
Mattha¨us Getlik,† Christian Gru¨tter,† Jeffrey R. Simard, Sabine Klu¨ter, Matthias Rabiller, Haridas B. Rode, Armin Robubi, and
Daniel Rauh*
Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, D-44227 Dortmund, Germany
ReceiVed March 8, 2009
The emergence of drug resistance remains a fundamental challenge in the development of kinase inhibitors
that are effective over long-term treatments. Allosteric inhibitors that bind to sites lying outside the highly
conserved ATP pocket are thought to be more selective than ATP-competitive inhibitors and may circumvent
some mechanisms of drug resistance. Crystal structures of type I and allosteric type III inhibitors in complex
with the tyrosine kinase cSrc allowed us to employ principles of structure-based design to develop these
scaffolds into potent type II kinase inhibitors. One of these compounds, 3c (RL46), disrupts FAK-mediated
focal adhesions in cancer cells via direct inhibition of cSrc. Details gleaned from crystal structures revealed
a key feature of a subset of these compounds, a surprising flexibility in the vicinity of the gatekeeper residue
that allows these compounds to overcome a dasatinib-resistant gatekeeper mutation emerging in cSrc.
Introduction
resistance to dasatinib. cSrc is known to be overexpressed or
up-regulated in several tumor types, most notably in glioblas-
toma, gastrointestinal, and prostate cancers, and represents a
target kinase for tumor therapy.8-11 In addition, our investigation
was stimulated by (a) type II kinase inhibitors such as lapatinib
and imatinib which not only bind in the ATP pocket but also
extend past the gatekeeper residue into a less conserved adjacent
allosteric site that is present solely in inactive kinase conforma-
tions and (b) a series of recently discovered type III inhibitors
that bind exclusively in the allosteric site of inactive cSrc without
making contact with the hinge region or the gatekeeper. Type
II inhibitors essentially stabilize this inactive conformation and
very often have slow dissociation rates, resulting in significantly
increased affinities over type I inhibitors.6 However, resistance
mutations are emerging at an increasingly rapid pace and often
limit the success of type I and type II inhibitors employed in
new targeted cancer therapies. The most common mutations
occur at the gatekeeper position in the hinge region12 in which
a small amino acid side chain (classically Thr) is exchanged
for a larger hydrophobic residue (Ile or Met). Such mutations
have been thoroughly investigated (breakpoint cluster region-
Abelson kinase (Bcr-Abl) (T315I), mast-stem cell growth factor
receptor kinase (c-KIT) (T670I), platelet derived growth fac-
tor receptor (PDGFRR) (T674I), and epidermal growth factor
receptor (EGFR) (T790M); cSrc (T338M) was previously used
Kinases and their associated signaling pathways are respon-
sible for the regulation of intracellular processes. Aberrant kinase
regulation can have a significant effect on the dynamics of these
intricate networks, ultimately resulting in total cellular disregu-
lation and contributing to the onset of several diseases including
cancer.1 On the basis of an improved understanding of kinase
malfunction in cancer biology, small organic molecules have
been developed for targeted cancer therapy. Although a dozen
kinase inhibitors are on the market and several more are in
clinical trials, inhibitor selectivity, lack of efficacy, and the
emergence of drug resistance remain fundamental challenges
in the development of kinase inhibitors that are effective in long-
term treatments.2-5 This can be attributed to the fact that most
kinase inhibitors are ATP-competitive molecules (type I inhibi-
tors), such as staurosporine and the dual specific Src/Abla
inhibitor dasatinib, which bind in the highly conserved ATP
pocket and form a critical hydrogen bond with the hinge region
of the kinase domain. Therefore, allosteric inhibitors that bind
to less conserved sites outside the ATP pocket would be
expected to have improved selectivity profiles and offer new
opportunities for scaffold development.6
We sought to use structure-based design principles to develop
organic molecules that stabilize an enzymatically inactive
conformation of the cSrc kinase domain while maintaining
potency against a particular mutation (T338M) at the gatekeeper
residue, an amino acid situated at the back of the ATP pocket
that is well-known for influencing type I inhibitor affinity and
selectivity profiles among kinases,7 of cSrc which results in
as a model system relevant to other drug resistant kinases13,14
)
and shown to cause a steric clash that impedes the binding of
ATP-competitive inhibitors, increase enzymatic activity, or
increase the affinity for ATP of some kinases.15-17 Given the
high mutation rate of tumor cells, the treatment of patients with
reversible inhibitors likely selects for pre-existing cancer cell
lines expressing the drug-resistant kinases. However, the
example of the Aurora kinase inhibitor VX-680, which retains
weak activity against drug-resistant T315I Bcr-Abl kinase
mutant18,19 (PDB code 2F4J) by extending out and away from
the hinge region and circumventing the gatekeeper residue,
shows that in principle reversible inhibitors can be obtained that
overcome drug resistance. It would therefore be desirable to
identify new chemical principles that can bind “around” these
mutations or target the kinase outside the ATP pocket and lock
it in an enzymatically inactive conformation.
#
Atomic coordinates and structure factors for cocrystal structures of
compound 3b in complex with cSrc wild type and drug resistant cSrc-
T338M can be accessed using PDB codes 3F3V and 3F3W, respectively.
cSrc wild type in complex with the drug dasatinib can be accessed using
the PDB code 3G5D.
* To whom correspondence should be addressed. Phone: +49 (0)231-
9742 6480. Fax: +49 (0)231-9742 6479. E-mail: daniel.rauh@cgc.mpg.de.
†
M.G. and C.G. contributed equally to this work.
a Abbreviations: Bcr-Abl, breakpoint cluster region-Abelson kinase;
c-KIT, mast-stem cell growth factor receptor kinase; PDGFRR, platelet
derived growth factor receptor; EGFR, epidermal growth factor receptor;
Abl, Abelson kinase; VEGFR, vascular endothelial growth factor receptor;
FAK, focal adhesion kinase; CDK2, cyclin-dependent kinase 2; PI3K,
phosphoinositide 3-kinase; TK, tyrosine kinase.
10.1021/jm9002928 CCC: $40.75  2009 American Chemical Society
Published on Web 05/22/2009

3916 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Table 1. Type I and Type III Inhibitors of cSrc and p38Ra
Getlik et al.
IC50 (µM)
KD (µM)
compd
1a
1b
2a
dasatinib
cSrc (wild type)
cSrc (T338M)
cSrc (wild type)
p38R
32.1 ( 7.520
64.1 ( 15.320
6.414
27.8 ( 10.2
nb
35 ( 0.9%20 at 50 µM
26 ( 1.3%20 at 50 µM
45 ( 1.3%20 at 10 µM
0.011 ( 0.003
0.05520 ( 0.005
0.01220 ( 0.002
nm*
nb14
0.0004 ( 0.0002
0.480 ( 0.40
0.495 ( 0.128*
a Structures of pyrazoloureas 1a, 1b, and 4-aminoquinazoline 2a are shown. IC50 values for inhibited enzyme activity (in µM) for a panel of inhibitors
against wild type (no drug resistance mutation at the gatekeeper) and drug resistant cSrc (cSrc-T338M). Kd values (in µM) for the same panel of pyrazoloureas
in cSrc and p38R. Pyrazoloureas 1a, 1b are potent inhibitors of p38R. Inhibitor 1a demonstrates balanced inhibition of wild type and drug resistant cSrc-
T338M, while bulky naphthyl derivative 1b weakly inhibits cSrc but fails to inhibit cSrc-T338M most likely because of a steric clash with the larger
gatekeeper residue (Supporting Information Figure 2). Quinazoline 2a was weakly sensed by acrylodan-labeled p38R but was strongly detected by acrylodan-
labeled cSrc as a weak type I inhibitor that binds to the hinge region of the kinase. The large bromophenyl moiety clashes with the gatekeeper side chain
in drug resistant cSrc-T338M and results in significant drop in affinity.14 The asterisk “*” denotes compounds for which Kd values were not measureable
(nm) because of high interference by intrinsic compound fluorescence. Acrylodan-labeled p38R exhibits an insensitivity to type I binders, unlike fluorescent
cSrc, while both fluorescent kinases serve as excellent sensors for DFG-out binders.
The above-mentioned allosteric site is formed when the
activation loop, a crucial structural component of the substrate
binding cleft participating in the recognition of substrates and
influencing the arrangement of the catalytic residues, adopts the
DFG-out conformation that is characteristic of inactive kinases.
In this conformation, the Phe of the DFG motif partially blocks
the ATP binding site, leaving the allosteric site available for
ligand binding. The equilibrium between the active DFG-in and
inactive DFG-out conformation can be modulated by phospho-
rylation of the activation loop, by a variety of protein-protein
interactions, or by the binding of type I, II, or III inhibitors to
particular protein conformations.
of cellular focal adhesions mediated by a substrate of cSrc, focal
adhesion kinase (FAK).
Results
Type III Inhibitors Active on Drug Resistant cSrc. We
recently developed a novel assay system that detects the binding
of type II and type III allosteric kinase modulators of the tyrosine
kinase cSrc by directly sensing the conformational changes that
they induce in the activation loop of the kinase. To achieve
this, we introduced the point mutation L407C into the activation
loop of the kinase to allow subsequent labeling with acrylodan,
a fluorophore sensitive to polarity changes of its environment.20
Conformational changes associated with the binding of allosteric
inhibitors bring the fluorophore toward the cleft between the C
and N lobes of the kinase, thereby modifying its fluorescence
characteristics. In a screening campaign, we employed this
newly developed cSrc assay to identify pyrazolourea compounds
(1a,b) as type III allosteric binders to cSrc with Kd values in
the micromolar range (Table 1). Although the binding of type
III inhibitors had not been reported previously for cSrc kinase,
several pyrazoloureas are known to be potent type III binders
of the serine/threonine kinase p38R MAP kinase with affinities
in the low nanomolar range.21,22 Enzyme activity assays were
subsequently used to confirm inhibition of cSrc kinase activity
in the micromolar range (Table 1), and the proposed type III
allosteric binding mode was confirmed by protein X-ray
crystallography (PDB codes 3F3U and 3F3T). Additionally,
these complex structures shed some light on the preference of
cSrc for the shared R2 N′-aryl moiety in 1a,b and highlight that
the size and degree of hydrophobicity of these aryl substituents
are important determinants for more energetically favorable
binding to inactive cSrc kinase conformations. We carried out
the same activity assay using the drug resistant cSrc variant
(T338M)13,14 and found that the presence of a bulkier gatekeeper
residue had no effect on 1a potency when compared to wild
type cSrc while 1b appeared to no longer be active against cSrc.
This further highlighting the importance of the urea-
anilino moiety of 1a in contributing to its affinity to cSrc, unlike
type I inhibitors such as quinazoline 2a and the aminothiazole
dasatinib, which show a dramatic loss in potency in cSrc-T338M
(Table 1). Similar observations have also been reported for
The recent availability of structural information of inactive
kinase conformations (p38R, EGFR, Abelson kinase (Abl),
vascular endothelial growth factor receptor (VEGFR), and
B-RAF) in complex with type II or type III inhibitors has
intensified the search for novel inhibitor scaffolds and stimulated
us to develop a direct binding assay that allowed for the first
time the unambiguous identification and discrimination of
organic molecules that target kinases of interest outside the ATP
pocket and stabilize the kinase domain in an enzymatically
inactive conformation.20 This new assay system reports on
structural changes of the activation loop associated with ligand
binding and enabled us to discover a series of pyrazoloureas as
truly allosteric binders (type III inhibitors) to the tyrosine kinase
cSrc. We employed protein X-ray crystallography to confirm
that these compounds bind to the DFG-out conformation and
lock the kinase in its inactive state. Here, we investigate the
pharmacological relevance of targeting this site in cSrc by using
principles of fragment-based drug design to systematically
develop a series of type II inhibitors that potently inhibit cSrc
in the low nanomolar range. We further demonstrate through
kinetic and structural analysis that a subset of these inhibitors
(3a-c) has surprising structural plasticity and is capable of
overcoming the emerging mechanism of gatekeeper-associated
drug resistance in kinases. We also demonstrated that one such
molecule, 3c, blocks proliferation of cancer cells by directly
inhibiting the tyrosine kinase cSrc and disrupting the formation

Hybrid Compound Design
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3917
Figure 1. Structure-based design of potent type II hybrid inhibitors of cSrc kinase. (a) The cocrystal structures of cSrc in complex with a pyrazolourea
type III inhibitor (PDB entry 3F3U)20 (gray) aligned with the structure of cSrc in complex with an ATP-competitive 4-aminoquinazoline (green)
(PDB entry 2QLQ)14 provided the rationale for structure-based drug design. The quinazoline core binds to the hinge region of the kinase, while the
pyrazolourea exclusively binds to the allosteric site of the kinase. The planes of the phenyl moieties of both inhibitors align adjacent to the Thr338
gatekeeper residue in chicken cSrc. (b) Rationally designed type II inhibitors based on the binding modes of type I 4-aminoquinazolines and type
III pyrazoloureas bound to cSrc. According to the notion of fragment-based drug discovery,25,26 chemically combining the two weak binders should
result in significantly higher binding affinities.
EGFR, where the T790M gatekeeper mutation results in
resistance to the marked drugs gefitinib and erlotinib.23 To better
understand the mechanism of dasatinib resistance in cSrc, we
solved its crystal structure in complex with wild type cSrc and
modeled the found binding mode to apo cSrc-T338M (PDB code
2QI8) (see Supporting Information Figure 1 and Supporting
Information methods). The alignment clearly shows that the
larger Met side chain of the mutant variant not only eliminates
an essential hydrogen bond of the inhibitor to the gatekeeper
side chain (T338) but also sterically impedes inhibitor binding.
The same steric repulsion by the larger gatekeeper residue is
likely responsible for the loss of activity of 1b and 2a in cSrc-
T338M (see Supporting Information Figure 2) and highlight that
a larger gatekeeper residue is not expected to interfere with
compounds that have the optimal size and degree of hydropho-
bicity to bind behind the gatekeeper position and exclusively
within the allosteric pocket of inactive kinase conformations
(DFG-out) such as 1a.
Design of Potent Type II Hybrid Inhibitors for cSrc
Kinase. Given the moderate micromolar IC50 values of the type
III pyrazoloureas 1a,b in cSrc, we set out to design more potent
type II cSrc inhibitors. We superimposed one of our cSrc-
pyrazolourea complexes (PDB code 3F3U) with one of our
recently solved cSrc structures in complex with a 4-amino-
quinazoline14 (PDB code 2QLQ) and found that the phenyl
substituents of both inhibitor scaffolds (aniline of the quinazoline
and aniline of the pyrazolourea) nicely align near the Thr338
gatekeeper side chain (Figure 1a), suggesting that a more potent
inhibitor could be generated by fusing both scaffolds via a 1,4-
or 1,3-substituted linkage (Figure 1b). Similar pyrazoloureas
fused to various hinge region binders are claimed in patent
literature to inhibit TIE-2 and RAF-kinase activity.24 In our
investigations, we were stimulated by both fragment-based
design approaches (where molecule fragments identified by
NMR25 or protein X-ray crystallography26,27 can be efficiently
linked or grown to generate molecules with increased affinity)
and the emerging concepts of the rational design of DFG-out
binders.6,28 Both methods have been proven to be powerful in
kinase lead discovery projects.29,30 Since pyrazoloureas have
been shown to be privileged motifs for the allosteric inhibition
of p38R and bind behind the mutation-prone gatekeeper residue,
we wanted to employ these scaffolds as starting points for
structure-guided design processes that take into account larger
gatekeeper side chains. We docked the proposed 1,4-para and
1,3-meta hybrid compounds into a published structure of BIRB-
796 bound to the DFG-out conformation of p38R22 and observed
different binding site geometries in the vicinity of the gatekeeper
residue and the hinge region in p38R when compared to inactive
cSrc (see Supporting Information Figure 3). Given these
observations, we predicted that cSrc would better accommodate
hybrid compounds fused via a 1,4-para linkage while a 1,3-
meta linkage should favor binding to p38R. More importantly,
we predicted that the 1,4-substitution pattern in these compounds
would provide the optimal geometry to avoid steric clashes with
the larger amino acid side chains found at the gatekeeper
position of drug resistant kinases. Although the 4-amino-

3918 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Table 2. Focused Library of Rationally Designed Type II Inhibitorsa
Getlik et al.
phosphotransfer. We observed a significant (up to 4 orders of
magnitude) increase in potency in the measured IC50 values
of these compounds when compared to the pyrazolourea (1a,
1b) or quinazoline (2a) moieties that were used to construct
each type II hybrid. The absolute values of the measured Kd
values are slightly higher when compared to the IC50 values
but follow the same trends for 1,4- and 1,3-substituted hybrid
compounds. Last and most important, the kinetics clearly
demonstrate that the 1,4-substituted hybrid compounds (3a-c)
show no loss of potency in the drug resistant cSrc-T338M
mutant in vitro.
Complex Crystal Structures of Novel Type II Inhibitors
in cSrc and Drug Resistant cSrc-T338M Mutant Variant.
To get deeper insights into the binding mode of this class of
type II inhibitors and to understand how these 1,4-substituted
inhibitors can overcome a bulky Met gatekeeper residue to
inhibit kinase activity without penalty of their affinity, we
cocrystallized cSrc (wild type and drug resistant T338M) with
RL45 (3b) and found that the compound indeed binds to the
DFG-out conformation and adopts the proposed type II inhibitor
binding mode that spans from the allosteric site into the distal
ATP binding pocket (Figure 2). The N1 of the quinazoline
moiety makes direct hydrogen bonding interactions with the
hinge region (M341) of the kinase, which is typically observed
for quinazoline binding to cSrc,14 cyclin-dependent kinase 2
(CDK2),31 p38R,31 Aurora,32 and EGFR.33,34 The pyrazolourea
moiety resides in the allosteric site and forms identical hydrogen
bonding interactions with helix C and the N-terminal region of
the activation loop (DFG-motif) as seen for the cSrc-type III
inhibitor complexes (PDB codes 3F3U and 3F3T)20 and as
observed in the structure of BIRB-796 complexed to p38R.22
The central phenyl ring of 3b that bridges the quinazoline and
pyrazolourea scaffolds is sandwiched between the gatekeeper
residue and the side chain of F405 of the DFG motif.
Interestingly, in the cSrc-T338M-3b complex the presence of
the sterically demanding Met gatekeeper forces the central
phenyl moiety of the inhibitor to rotate 90° to avoid the steric
clash with the amino acid side chain such that the plane of the
phenyl ring of the inhibitor now faces Cε of M338. The side
chain of F405 also rotates by 90° to conserve the electrostatically
favorable edge-to-face orientation35 of both π-electron systems
(inhibitor phenyl and phenyl side chain of F405) (Figure 2c
and Figure 2d), suggesting an additional stabilizing role for this
interaction. Rotation of the central phenyl element in 1,3-
substituted hybrid compounds 3d and 3e is not possible without
disrupting the binding orientation of either the quinazoline or
pyrazolourea moiety with the protein and provides the explana-
tion for why 1,3-disubstituted hybrids such as 3d and 3e do
not bind to drug resistant cSrc-T338M (Figure 3). In a recent
study, Shokat and colleagues report on a similar flexibility of a
pyrazolopyrimidine based type I inhibitor to be able to accom-
modate either wild type cSrc or a phosphoinositide 3-kinase
(PI3K) (Ile at the gatekeeper) to make a dual specific tyrosine
kinase (TK)/PI3K inhibitor.7
IC50 (µM)
Kd (µM)
compd cSrc (wild type) cSrc (T338M) cSrc (wild type)
p38R
3a
3b
3c
3d
3e
0.071 ( 0.010 0.101 ( 0.004
0.021 ( 0.005 0.034 ( 0.012
0.014 ( 0.001 0.023 ( 0.004
0.207 ( 0.079 nb
0.174 ( 0.038 0.127 ( 0.026
0.073 ( 0.016 0.342 ( 0.039
0.056 ( 0.013 0.245 ( 0.055
0.256 ( 0.064 0.050 ( 0.016
0.239 ( 0.045 0.074 ( 0.002
0.235 ( 0.094 36.8 ( 6
a Structures of 1,4-para (3a-c) and 1,3-meta (3d,e) quinazoline-pyrazolo-
urea hybrid compounds are shown. IC50 and Kd values (in µM) for a panel
of inhibitors against wild type cSrc, drug resistant cSrc-T338M, and p38R.
1,4-Substituted hybrids show the best balance of potency and selectivity
for cSrc (wild type and drug resistant). The R1 substituents in position 6 of
the quinazoline core are important determinants for potency in cSrc and
render a clear SAR with 3c being the most potent hybrid compound for
both cSrc wild type and drug resistant cSrc-T338M. 1,3-Substituted inhibitor
cores direct selectivity of (3d,e) toward p38R and significantly decreases
affinity to drug resistant cSrc-T338M.
quinazoline 2a and the identified pyrazoloureas 1a and 1b are
alone weak inhibitor fragments with IC50 values in the micro-
molar range in wild type cSrc, we expected that the resulting
1,4-substituted hybrid compounds would show significantly
increased potency in inhibiting not only wild type but also the
otherwise drug resistant cSrc-T338M mutant variant.
Synthesis of a Focused Library of 4-Aminopyrazol-
oureaquinazolines as Novel Type II Inhibitors. We synthe-
sized a small focused library of fused quinazoline-pyrazolourea
hybrids as potent type II inhibitors of cSrc (Table 2 and Scheme
1). The panel included analogues with varying inhibitor
geometries designed to orient around the sterically bulky
gatekeeper residue of drug resistant cSrc-T338M or to prefer-
entially bind to p38R, a kinase that is known to be inhibited
potently by compounds containing the pyrazolourea scaffold.
Detailed synthetic procedures are described in the Experimental
Section.
In Vitro Characterization of Novel Type II Hybrid cSrc
Inhibitors. To test whether the allosteric site in cSrc confirmed
by our previous cocrystallization experiments is indeed drug-
gable in solution and to test the above-mentioned hypotheses
regarding inhibitor selectivity to p38R and drug resistant cSrc-
T338M, we first measured the Kd of each hybrid compound
using the fluorescent-labeled kinase binding assay described
elsewhere.20 The binding data obtained from each fluorescent
kinase confirmed the expected binding preference of 1,4- and
1,3-substituted hybrid compounds for cSrc and p38R, respec-
tively. Additionally, we performed enzyme activity assays for
cSrc (wild type and drug resistant T338M) using several type
II hybrid compounds (Table 2) to confirm inhibition of
Type II cSrc Inhibitors Disrupt Cell-to-Cell Contacts in
cSrc-Dependent Cancer Cell Lines. To assess cSrc inhibition
by the most potent 1,4-substituted hybrid 3c in cellular systems,
we treated PC3 and DU145 prostate carcinoma cell lines10 with
different concentrations of 3c, 100 nM dasatinib (positive Src
inhibition control), or vehicle (DMSO). We monitored the
phosphorylation state of Y416 (an autophosphorylation site in
the activation loop of cSrc) and Y576/Y577 (two residues in
the activation loop of FAK that are phosphorylated by cSrc to
fully activate FAK). FAK is a nonreceptor tyrosine kinase that

Hybrid Compound Design
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3919
Scheme 1. Synthesis of Quinazoline Derivativesa
a Reagents and conditions: (a) (i) formamidine acetate, 2-methoxyethanol, 132 °C; (ii) SOCl2, cat. DMF, reflux; (b) aminophenylpyrazolourea hydrochlorides,
DIPEA, CH2Cl2, room temp; (c) Pd/C, ammonium formate, EtOH, reflux; (d) 3b, propionyl chloride, DIPEA, THF, 0 °C.
localizes to focal adhesions that form between cells and is a
key regulator of cell cycle progression, cell survival, and cell
migration.36 Its activation by cSrc results in the disruption of
focal adhesions, causing loss of cell-cell and cell-matrix
contacts and apoptosis.11,37 The overexpression of FAK and cSrc
has been shown to lead to increased cell invasion and metastasis
in both breast and colon cancers.38,39
After 5 h of treatment with dasatinib or 3c, confluent PC3
and DU145 cells exhibited markedly reduced pSrc (Y416) and
pFAK (Y576/Y577) levels (Figure 4a) as well as a loss of cell
adhesions and a significant reduction in the number of cells
(Figure 4b). Given the reduction of pFAK, this change in cellular
morphology is associated with direct inhibition of Src kinase
by 3c via a reduction in downstream signaling through FAK.
Interestingly, we also observed an up-regulation of Src expres-
sion in both cell lines with increasing 3c concentration. A
positive feedback mechanism involving the up-regulation of Src
expression in response to inhibited Src activity has also been
observed elsewhere,40 further suggesting that 3c shows selectiv-
ity toward Src kinase in vivo. In order to determine kinase
selectivity for our newly developed type II hybrid compounds,
kinase profiling was performed for 3b and 3e against a selected
panel of 27 different kinases at a concentration of 5 µM (Ambit
Biosciences) (see Supporting Information Figure 4).
The combination of in vitro binding and activity assays
demonstrates that the quinazoline-pyrazolourea hybrids pre-
sented here are promising kinase inhibitor scaffolds for further
medicinal chemistry initiatives. The gatekeeper is a Thr in many
tyrosine kinases and also serves as a crucial determinant of type
I inhibitor selectivity and affinity. Therefore, the development
of these type II hybrid inhibitors combined with the observa-
tion of a potential cross-talk between the inhibitor and the side
chains of the drug resistant hydrophobic gatekeeper and/or the
DFG phenylalanine residue provides an attractive chemical
biological strategy for developing potent inhibitors that can
selectively target such kinases while also overcoming the
increasingly common gatekeeper mutation-associated drug
resistance.
inhibitors that are effective in long-term treatments. Here, we
present a series of type III inhibitors active on a dasatinib
resistant cSrc-T338M mutant variant. Despite weak binding to
cSrc in comparison to p38R, these pyrazoloureas served as an
excellent starting point for the development of these more potent
cSrc inhibitors. On the basis of the analysis of crystal structures
of cSrc in complex with different type III pyrazoloureas or with
type I quinazoline-based inhibitors, we designed quinazoline-
pyrazolourea type II hybrid inhibitors that proved to be excellent
cSrc inhibitors. Several derivatives were synthesized to explore
SAR and confirmed our prediction that the geometry of these
compounds would (i) govern their preferential binding to either
cSrc or p38R and (ii) permit the circumvention of larger
gatekeeper residues that are known to commonly cause drug
resistance in certain cancer cell lines. We were able to confirm
these hypotheses by using direct Kd determination (acrylodan-
labeled cSrc and p38R), kinase activity assays, and protein X-ray
crystallography. The increased affinity of these type II hybrid
compounds was not only due to the added 4-aminoquinazoline
moiety to make contact with the hinge region of the kinase but
also due to the substitution pattern of the central phenyl moiety
that is positioned near the gatekeeper residue. Although both
1,4-para and 1,3-meta hybrid compounds inhibit cSrc in the low
nanomolar to mid-nanomolar range, only the central phenyl of
the 1,4-substituted hybrid overcame the T338M cSrc drug
resistance mutation by having the rotational freedom necessary
to avoid a clash with the bulkier gatekeeper side chain without
also disturbing the binding interactions formed by the rest of
the drug molecule. In a recent study, Shokat and colleagues
report on a similar flexibility observed in a pyrazolopyrimidine-
based type I inhibitor that is accommodated by either wild type
cSrc (gatekeeper Thr) or a PI3-kinase (gatekeeper Ile) to serve
as a dual-specificity TK/PI3K inhibitor.7 In a second paper by
the same group, the authors report on the design of a series of
pyrazolopyrimidinoureas as potent type II cSrc inhibitors.41
Complex structures with cSrc revealed a para-substituted phenyl
ring of the inhibitors in proximity to the gatekeeper. The authors
conclude that the relative orientation of this phenyl ring to the
o-methylphenylamino moiety of imatinib, which is responsible
for drug resistance in Abl-T315I, may suggest that these
inhibitors can bind to Abl-T315I. The activity of our type II
inhibitors in cSrc-relevant prostate cancer cell lines supports
Discussion and Conclusion
Inhibitor selectivity and the emergence of drug resistance
remain fundamental challenges in the development of kinase

3920 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Getlik et al.
Figure 2. 1,4-Substituted hybrid compound (3b) in complex with wild type cSrc and drug resistant cSrc-T338M reveals inhibitor flexibility for
overcoming a gatekeeper-associated drug resistance mutation. Stereodiagrams of the experimental electron densities (ligand red, protein gray) of
cSrc-3b (a) and cSrc-T338M-3b (b) both at 2.6 Å resolutions are shown (2Fo - Fc map contoured at 1σ). Hydrogen bonding interactions of the
inhibitors with helix C (blue), the DFG-motif (orange), and the hinge region (pink) are shown by red dotted lines. The kinase domain is in the
inactive conformation, and the pyrazolourea moiety resides in the allosteric site flanked by helix C and the DFG-motif. N1 of the quinazoline
makes a direct hydrogen bond to the main chain amide of M341, which is an interaction commonly formed between anilinoquinazolines and
the hinge region of several other protein kinase domains. In both complexes, the central phenyl moiety that links the quinazoline scaffold with the
pyrazolourea fragment interacts with the side chain of F405 (DFG motif) in a favorable edge-to-face orientation. (c) van der Waals radii of the
inhibitor (mesh), the gatekeeper residues T338/M338 (pink spheres), and the side chain of F403 (orange spheres) explain conformational changes
of the central phenyl moiety of the inhibitor to bypass steric clashes with the side chain of M338, allowing 3b to bind to drug resistant cSrc-T338M.
(d) A larger side chain at the gatekeeper position results in a 90° flip of the central phenyl moiety of the inhibitor. Likewise, the side chain of F405
is rotated by 90° to maintain the electrostatically favorable edge-to-face orientation of both π-electron systems conserved.35
the structure-based rationale used in the design of these more
potent hybrid compounds. It is believed that the in vivo efficacy
of drugs against their primary targets is strongly correlated to
the drug-target residence time, the concept that a drug remains
efficacious only as long as it is bound to its target.42 In the
case of cSrc, we were able to use our acrylodan-labeled cSrc
assay to confirm that the 1,4-substituted compounds (3a-c)
dissociate more slowly than the less potent 1,3-substituted
compounds (3d, 3e) while the binding rates for all compounds
were essentially the same (see Supporting Information Table
2). Although it is not clear which kinases will develop point-
mutation-associated drug resistance under the regime of targeted
therapies, it is evident that this is likely to become a major
problem in the future as more kinase inhibitors are used to treat
larger patient populations. As gleaned from the emergence of
resistance to antimicrobial or antiviral agents by way of chemical
inactivation of essential proteins and selective pressures that
increase the incidence of mutations that convey said resistance,
cancer cells carrying these mutations will also become more
pronounced in rapidly dividing cell populations. To account for
this challenge and to stimulate the design of future generation
kinase inhibitors, extensive investigations are underway to

Hybrid Compound Design
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3921
Figure 3. 1,3-Substituted hybrid (3e) docked to drug resistant cSrc-T338M. The 1,3-meta hybrid compound 3e (green) was docked manually into
wild type cSrc-3b (a) and drug resistant cSrc-T338M-3b (b) complexes. Care was taken to conserve the essential hydrogen bonding interaction of
the quinazoline N1 with the backbone of the hinge region and occupation of the allosteric site by the pyrazolourea moiety. The inhibitor adopts a
binding mode well tolerated by a small gatekeeper residue (T338). (c) In drug resistant cSrc-T338M, the central 1,4-substituted phenyl element of
3a-c can freely rotate to adapt to a larger gatekeeper residue. (d) Free rotation of this crucial element in 1,3-substituted hybrid compounds is not
possible and would result in either loss of the backbone hydrogen bond or displacement of the pyrazolourea from the allosteric site to accommodate
the larger gatekeeper. Decreased inhibitor flexibility helps to explain why binding of 3d and 3e to drug resistant cSrc-T338M is significantly
compromised.
at 400 and 101 MHz, respectively. 1H chemical shifts are reported
provoke drug resistance formation in model organisms treated
in δ (ppm) as s (singlet), d (doublet), dd (doublet of doublet), t
(triplet), q (quartet), m (multiplet), and bs (broad singlet) and are
referenced to the residual solvent signal: CDCl3 (7.26), DMSO-d6
(2.50). 13C spectra are referenced to the residual solvent signal:
CDCl3 (77.0) or DMSO-d6 (39.0). All final compounds were
purified to >95% purity, as determined by high-performance liquid
chromatography (HPLC) (Supporting Information Table 3). Purity
was measured using Agilent 1200 series HPLC systems with UV
detection at 210 nm (system: Agilent Eclipse XDB-C18 4.6 mm
× 150 mm, 5 µM, 10-100% CH3CN in H2O, with 0.1% TFA, for
15 min at 1.0 mL/min). High resolution electrospray ionization mass
spectra (ESI-FTMS) were recorded on a Thermo LTQ Orbitrap
(high resolution mass spectrometer from Thermo Electron) coupled
to an “Accela” HPLC system supplied with a “Hypersil GOLD”
column (Thermo Electron). Analytical TLC was carried out on
Merck 60 F245 aluminum-backed silica gel plates. Compounds
were purified by column chromatography using Baker silica gel
(40-70 µm particle size). Preparative HPLC was conducted on
a Varian HPLC system (Pro Star 215) with a VP 250/21 nucleosil
C18 PPN column from Macherey-Nagel and monitored by UV
at λ ) 254 nm.
with specific kinase inhibitors with the hope of uncovering future
clinically relevant mutant kinase alleles to be used as predictive
markers. Such knowledge will advance the concept of personal-
ized cancer therapies by using the compounds best suited for
the identified tumor cell type.43-45 In an alternative approach,
knowledge about which position(s) is likely to develop relevant
drug resistance mutations in kinases will be crucial for the design
of next generation drugs that can overcome them. Although
kinases remain one of the largest classes of enzymes studied,
strategies for overcoming drug resistance are a challenging task
and innovative solutions still need to be found. For example,
Crespo et al.46 showed that imatinib can be re-engineered to
minimize the entropic cost of binding to drug resistant D816V
Abl mutant by promoting disorder in the DFG loop. Our results
illustrate a powerful alternative rationale to overcome drug
resistance by generating type II inhibitors that have the intrinsic
ability to adapt to the binding site distortions induced by these
mutations while also locking the kinase in an inactive
conformation.
3-tert-Butyl-1-m-tolyl-1H-pyrazol-5-amine (4a).47 To a solution
of m-tolylhydrazine (0.5 g, 3.96 mmol) and 4,4-dimethyl-3-
oxopentanenitrile (0.49 g, 3.96 mmol) in EtOH (10 mL) was added
concentrated HCl (2 mL), and the reaction mixture was refluxed at
90 °C for 24 h. The volatiles were removed in vacuo, and water
was added and extracted with DCM (3 × 30 mL). The combined
organic layers were washed with saturated NaHCO3 (1 × 30 mL)
Experimental Section
Chemistry. Unless otherwise noted, all reagents and solvents
were purchased from Acros, Fluka, Sigma, Aldrich, or Merck and
used without further purification. Dry solvents were purchased as
1
anhydrous reagents from commercial suppliers. H and 13C NMR
spectra were recorded on a Bruker Avance DRX 400 spectrometer

3922 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Getlik et al.
Figure 4. Reduction of cell-to-cell-contacts and cell proliferation in PC3 and DU145 cells by 3c. PC3 (a) and DU145 (b) cells were treated for
5 h with 3c (1, 2, 5, and 10 µM), dasatinib (100 nM), or vehicle (DMSO). Cells were lysed and blotted for indicated proteins. pSrc and pFAK levels
are markedly reduced in response to treatment with 3c and dasatinib (left panel). Total expression of FAK was unchanged, while cSrc expression
was increased in both cell lines. Cell-to-cell contacts visualized by light microscopy at 10× magnification. PC3 and DU145 cells show markedly
reduced cell-to-cell-contacts and fewer intact cells after treatment with 3c (10 µM) or dasatinib (100 nM).
and dried over Na2SO4. Evaporation of the volatiles yielded the
crude which was recrystallized from petrol ether/MeOH leading to
668 mg (74%) of colorless crystals: H NMR (400 MHz, CDCl3)
stirred at room temperature for 10 min before 4a was added in one
portion. The reaction mixture was heated to 60 °C for 3 h. After
cooling to room temperature, the mixture was portioned between
EtOAc and H2O. The aqueous layer was extracted with EtOAc (3
× 10 mL). The combined organic layer was washed with brine
and H2O, dried over Na2SO4, and evaporated in vacuo. A solution
of the obtained solid and HCl (4 M solution in dioxane) in dioxane
was stirred at room temperature for 30 min before the volatiles
were removed in vacuo. The product was used without further
purification.
1
δ 7.34 (s, 1H), 7.30 (d, J ) 4.7 Hz, 2H), 7.10 (t, J ) 3.6 Hz, 1H),
5.49 (s, 1H), 3.81 (s, 2H), 2.37 (s, 3H), 1.30 (s, 9H); 13C NMR
(101 MHz, CDCl3) δ 162.23, 145.11, 139.80, 138.50, 129.34,
128.18, 125.20, 121.22, 87.67, 32.44, 30.51, 21.60. HRMS (ESI-
MS) calcd: 230.165 17 for C14H20N3 [M + H+]. Found: 230.165 03.
2,2,2-Trichloroethyl 3-tert-Butyl-1-m-tolyl-1H-pyrazol-5-yl-
carbamate (5).48,49 A mixture of 4a (500 mg, 2.2 mmol), H2O (6
mL), EtOAc (10 mL), and NaOH (130 mg, 3.3 mmol) was stirred
at 0 °C for 30 min. Then 2,2,2-trichloroethyl chloroformate (440
µL, 3.3 mmol) was added dropwise. After a further 30 min the ice
bath was removed and the reaction mixture was stirred at room
temperature for 2 h. The organic layer was separated from the
aqueous layer which was extracted with EtOAc (4 × 10 mL). The
combined organic layers were washed with brine twice, dried over
Na2SO4, and evaporated in vacuo. The crude green solid was
recrystallized from hexane to yield 536 mg (61%) of colorless
1-(4-Aminophenyl)-3-(3-tert-butyl-1-m-tolyl-1H-pyrazol-5-
yl)urea Hydrochloride (7a). 7a was prepared as described above
in the general procedure using N-Boc-p-phenylenediamine (157 mg,
0.8 mmol), DMSO (2 mL), DIPEA (400 µL, 2.3 mmol), 5 (305
mg, 0.8 mmol). The crude material was purified on silica gel
(30-100% EtOAc/petrol ether) to yield 339 mg (96%) of the off-
white solid product tert-butyl 4-(3-(3-tert-butyl-1-m-tolyl-1H-
pyrazol-5-yl)ureido)phenylcarbamate (6a). Then 6a (339 mg, 0.7
mmol), HCl (4 mL of a 4 M solution in dioxane), and dioxane (1
mL) were used. Without further purification, an amount of 249 mg
1
crystals: H NMR (400 MHz, CDCl3) δ 7.35 (t, J ) 7.7 Hz, 1H),
7.27 (s, 1H), 7.21 (t, J ) 7.6 Hz, 2H), 6.87 (s, 1H), 6.40 (s, 1H),
4.79 (s, 2H), 2.39 (s, 3H), 1.33 (s, 9H); 13C NMR (101 MHz,
CDCl3) δ 162.52, 150.98, 140.32, 137.78, 134.97, 129.70, 129.37,
126.09, 121.99, 95.04, 75.05, 32.65, 30.51, 21.59. HRMS (ESI-
MS) calcd 404.063 93 for C17H21O2N335Cl3 and 406.066 44 for
C17H21O2N335Cl237Cl [M + H+]. Found: 404.069 00 and 406.065 56.
General Procedure for the Preparation of Aminophenyl-3-
(3-tert-butyl-1-tolyl-1H-pyrazol-5-yl)ureaHydrochlorides(7a,b).48,49
N-Boc-phenylenediamine was dissolved in dry DMSO and treated
with DIPEA. The reaction mixture was placed under argon and
1
(86%) of the off-white solid product 7a was obtained: H NMR
(400 MHz, DMSO-d6) δ 10.27 (bs, 2H), 9.78 (s, 1H), 8.83 (s, 1H),
7.51 (d, J ) 8.8 Hz, 2H), 7.35 (m, 5H), 7.21 (d, J ) 7.3 Hz, 1H),
6.37 (s, 1H), 2.37 (s, 3H), 1.28 (s, 9H); 13C NMR (101 MHz,
DMSO-d6) δ 160.54, 151.83, 139.44, 138.87, 138.08, 137.18,
129.06, 128.08, 125.14, 124.94, 123.86, 121.41, 118.72, 95.96,
32.06, 30.18, 20.99. HRMS (ESI-MS) calcd: 364.213 19 for
C21H26N5O [M + H+]. Found: 364.213 15.

Hybrid Compound Design
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3923
1-(3-Aminophenyl)-3-(3-tert-butyl-1-m-tolyl-1H-pyrazol-5-
yl)urea Hydrochloride (7b). 7b was prepared as described above
in the general procedure using N-Boc-m-phenylenediamine (92 mg,
0.4 mmol), DMSO (2 mL), DIPEA (150 µL, 0.9 mmol), 5 (180
mg, 0.6 mmol). The reaction mixture was heated to 60 °C for 24 h.
The crude material was purified on silica gel (30-50% EtOAc/
petrol ether), yielding 150 mg (74%) of the off-white solid product
tert-butyl 3-(3-(3-tert-butyl-1-m-tolyl-1H-pyrazol-5-yl)ureido)phe-
nyl carbamate (6b). Then 6b (150 mg, 0.3 mmol), HCl (3 mL of
a 4 M solution in dioxane), and dioxane (1 mL) were used. Without
further purification, an amount of 120 mg (92%) of the white
(s, 1H), 8.70 (s, 1H), 8.55 (dd, J ) 2.2, 9.2 Hz, 1H), 8.43 (s, 1H),
8.01 (s,1H), 7.93 (d, J ) 9.2 Hz, 1H), 7.47 (d, J ) 7.9 Hz, 1H),
7.43 (t, J ) 7.7 Hz, 1H), 7.32 (dd, J ) 7.5, 15.7 Hz, 3H), 7.24 (t,
J ) 6.9 Hz, 2H), 6.40 (s, 1H), 2.40 (s, 3H), 1.28 (s, 9H); 13C NMR
(101 MHz, DMSO-d6) δ 160.67, 158.86, 157.68, 153.11, 151.41,
144.52, 139.62, 138.94, 138.81, 138.39, 137.24, 129.49, 129.13,
128.82, 128.05, 126.64, 125.12, 121.56, 120.98, 116.81, 114.45,
114.38, 112.64, 94.73, 32.05, 30.24, 20.99. HRMS (ESI-MS) calcd:
537.235 71 for C29H29N8O3 [M + H+]. Found: 537.235 13.
General Procedure for the Preparation of (6-Aminoquinazo-
lin-4-ylamino)phenyl)-3-(3-tert-butyl-1-m-tolyl-1H-pyrazol-5-
yl)ureas (3b,e). A solution of 3a,d, ammonium formate, and Pd/C
in absolute EtOH was refluxed at 90 °C for 3 h. The reaction
mixture was filtered over Celite, and the volatiles were removed
in vacuo to yield the crude material which was purified on silica
gel (2-5% MeOH/DCM). The silica gel was neutralized using
DCM/5% Et3N. Further purification and characterization of each
derivative is described below.
1
product 7b was obtained: H NMR (400 MHz, DMSO-d6) δ 9.98
(s, 1H), 8.91 (s, 1H), 7.65 (s, 1H), 7.40-7.32 (m, 5H), 7.21 (d, J
) 7.35 Hz, 1H), 6.98 (m, 1H), 6.38 (s, 1H), 2.38 (s, 3H), 1.28 (s,
9H); 13C NMR (101 MHz, DMSO-d6) δ 160.55, 151.81, 140.76,
138.90, 138.06, 137.09, 132.08, 130.14, 129.08, 128.10, 124.91,
121.37, 117.26, 116.49, 112.44, 96.07, 32.07, 30.18, 21.01. HRMS
(ESI-MS) calcd: 364.213 19 for C21H26N5O [M + H+]. Found:
364.213 16.
1-(4-(6-Aminoquinazolin-4-ylamino)phenyl)-3-(3-tert-butyl-1-
m-tolyl-1H-pyrazol-5-yl)urea (3b). 3b was prepared as described
above in the general procedure using 3a (86 mg, 0.16 mmol),
ammonium formate (71 mg, 1.1 mmol), EtOH, and Pd/C. The
6-Nitroquinazolin-4-ol (11). A solution of 2-amino-5-nitroben-
zoic acid (9.5 g, 52 mmol) and formamidine acetate (22.0 g, 210
mmol) in 2-methoxyethanol (300 mL) was refluxed at 132 °C
overnight and then concentrated in vacuo. The precipitate was
filtered and washed with MeOH to yield 7.95 g (79%) of a brown
powder. The product was used without further purification: 1H NMR
(400 MHz, DMSO-d6) δ 12.70 (s, 1H), 8.71 (s, 1H), 8.48 (d, J )
7.5 Hz, 1H), 8.27 (s, 1H), 7.79 (d, J ) 8.4 Hz, 1H); 13C NMR
(101 MHz, DMSO-d6) δ 160.54, 152.89, 148.92, 144.88, 129.00,
128.18, 3c.64, 121.86. HRMS (ESI-MS) calcd: 192.040 37 for
C8H6N3O3 [M + H+]. Found: 192.040 18.
1
product was obtained as 80 mg (98%) of a yellow powder: H
NMR (400 MHz, DMSO-d6) δ 9.25 (s, 1H), 9.02 (s, 1H), 8.38
(s, 1H), 8.27 (s, 1H), 7.72 (d, J ) 9.0 Hz, 2H), 7.50 (d, J ) 8.8
Hz, 1H), 7.43 (d, J ) 7.7 Hz, 1H), 7.36 (m, 5H), 7.28 (d, J )
25.1 Hz, 1H), 7.22 (dd, J ) 2.3, 8.9 Hz, 1H), 6.38 (s, 1H), 5.51
(s, 2H), 2.40 (s, 3H), 1.29 (s, 9H); 13C NMR (101 MHz, DMSO-
d6) δ 160.64, 156.02, 151.57, 149.97, 147.13, 142.40, 138.89,
138.45, 137.36, 134.70, 134.34, 129.09, 128.57, 127.98, 125.05,
123.41, 3c.58, 121.50, 118.25, 116.57, 101.12, 94.88, 32.05,
30.24, 20.99. HRMS (ESI-MS) calcd: 507.261 53 for C29H31N8O
[M + H+]. Found: 507.260 84.
General Procedure for the Preparation of 1-(3-tert-Butyl-1-
m-tolyl-1H-pyrazol-5-yl)-3-((6-nitroquinazolinylamino)phenyl)-
ureas (3a,d). A two-neck flask was flushed with argon and charged
with 11 and thionyl chloride. A catalytic amount of DMF was
added, and the reaction mixture was refluxed at 78 °C overnight.
The thionyl chloride was evaporated under reduced pressure. The
residue was dissolved in DCM and filtered over silica gel to give
4-chloro-6-nitroquinazoline (12) which was dried under high
vacuum and used without further purification. A solution of
pyrazolourea hydrochloride 7a,b and DIPEA in DCM was stirred
for 10 min before 12 was added in one portion. The reaction mixture
was stirred at room temperature for 24 h. Saturated NaHCO3 was
added, and the organic layer was separated from the aqueous layer
which was then extracted with EtOAc (4 × 10 mL). The combined
organic layers were dried over Na2SO4 and the volatiles were
removed in vacuo, yielding an orange solid. Further purification
and characterization of each derivative are described below.
1-(3-tert-Butyl-1-m-tolyl-1H-pyrazol-5-yl)-3-(4-(6-nitroquinazo-
lin-4-ylamino)phenyl)urea (3a). 3a was prepared as described
above in the general procedure using 7a (100 mg, 0.3 mmol),
DIPEA (171 µL, 1 mmol), DCM (2 mL), and 10 (52 mg, 0.3 mmol).
The reaction mixture was stirred at room temperature for 24 h.
The crude was purified on silica gel (1-4% MeOH/DCM) to give
1-(3-(6-Aminoquinazolin-4-ylamino)phenyl)-3-(3-tert-butyl-1-
m-tolyl-1H-pyrazol-5-yl)urea (3e). 3e was prepared as described
above in the general procedure using 3d (50 mg, 0.09 mmol),
ammonium formate (41 mg, 0.7 mmol), EtOH (1.5 mL), and Pd/
C. The crude material was purified on silica gel (2-5% MeOH/
DCM). In a second purification step the product was purified by
HPLC (MeCN/H2O with 0.1% TFA) to yield 17 mg (36%) of the
1
free base as a green powder: H NMR (400 MHz, DMSO-d6) δ
9.31 (s, 1H), 9.08 (s, 1H), 8.40 (s, 1H), 8.31 (s, 1H), 7.98 (s, 1H),
7.52 (d, J ) 8.8 Hz, 1H), 7.38 (m, 5H), 7.23 (m, 4H), 6.40 (s, 1H),
5.57 (s, 2H), 2.39 (s, 3H), 1.28 (s, 9H); 13C NMR (101 MHz,
DMSO-d6) δ 160.66, 156.00, 151.36, 149.76, 147.14, 142.55,
140.38, 139.39, 138.93, 138.38, 137.34, 129.12, 128.62, 128.05,
125.14, 123.58, 121.58, 116.75, 116.55, 115.66, 112.78, 111.46,
101.16, 94.54, 32.04, 30.24, 20.98. HRMS (ESI-MS) calcd:
507.261 53 for C29H31N8O [M + H+]. Found: 507.260 83.
N-(4-(4-(3-(3-tert-Butyl-1-m-tolyl-1H-pyrazol-5-yl)ureido)phe-
nylamino)quinazolin-6-yl)propionamide (3c). A Schlenk tube was
flushed with argon and charged with 3b (15 mg, 29.61 µmol) and
dry THF (1 mL), and the mixture was cooled to 0 °C before DIPEA
(12.67 µL, 74.02 µmol) was added. A solution of propionyl chloride
was prepared using 10 µL of propionyl chloride in 10 mL of dry
THF. Then the solution of propionyl chloride (190 µL) was added
dropwise to the reaction mixture which was stirred for 10 min before
the ice bath was removed. After 1.5 h, an amount of 100 µL of the
propionyl chloride solution was added and the mixture was stirred
for a further 45 min until the reaction was complete. The mixture
was basified with triethylamine before brine and EtOAc were added.
The water phase was extracted with EtOAc (3 × 10 mL), and the
combined organic layers were washed with water and dried over
Na2SO4. The volatiles were removed in vacuo, leading to a yellow
solid which was purified on silica gel using DCM/1-4% MeOH.
An amount of 12 mg (72%) of the yellow solid product was
obtained: 1H NMR (400 MHz, DMSO-d6) δ 10.31 (s, 1H), 9.73 (s,
1H), 9.39 (bs, 1H), 8.89 (bs, 1H), 8.70 (s, 1H), 8.44 (s, 1H), 7.86
(d, J ) 9.0 Hz, 1H), 7.68 (dd, J ) 8.9, 16.8 Hz, 3H), 7.40 (m,
5H), 7.20 (d, J ) 6.6 Hz, 1H), 6.35 (s, 1H), 2.43 (m, 5H), 1.28 (s,
9H), 1.13 (t, J ) 7.5 Hz, 3H); 13C NMR (101 MHz, DMSO-d6) δ
1
98 mg (73%) of the orange solid product: H NMR (400 MHz,
DMSO-d6) δ 10.40 (s, 1H), 9.65 (d, J ) 2.4 Hz, 1H), 9.11 (s, 1H),
8.67 (s, 1H), 8.54 (dd, J ) 2.4, 9.2 Hz, 1H), 8.41 (s, 1H), 7.91 (d,
J ) 9.1 Hz, 1H), 7.72 (d, J ) 8.9 Hz, 2H), 7.46 (m, 2H), 7.42 (d,
J ) 7.7 Hz, 1H), 7.34 (m, 2H), 7.24 (d, J ) 7.43 Hz, 1H), 6.39 (s,
1H), 2.40 (s, 3H), 1.29 (s, 9H); 13C NMR (101 MHz, DMSO-d6) δ
160.11, 158.70, 157.86, 153.13, 151.54, 144.43, 138.91,138.43,
137.26, 136.17, 132.59, 129.42, 129.11, 128.01, 126.57, 125.06,
123.69, 121.51, 120.85, 118.12, 114.39, 94.95, 32.05, 30.24, 20.99.
HRMS (ESI-MS) calcd: 537.235 71 for C29H29N8O3 [M + H+].
Found: 537.235 18.
1-(3-tert-Butyl-1-m-tolyl-1H-pyrazol-5-yl)-3-(3-(6-nitroquinazo-
lin-4-ylamino)phenyl)urea (3d). 3d was prepared as described
above in the general procedure using 7b (120 mg, 0.3 mmol),
DIPEA (154 µL, 0.9 mmol), DCM (2 mL), and 10 (63 mg, 0.3
mmol). The crude was purified on silica gel (1-4% MeOH/DCM)
1
to give 78 mg (49%) of the orange solid product: H NMR (400
MHz, DMSO-d6) δ 10.44 (s, 1H), 9.68 (d, J ) 2.1 Hz, 1H), 9.17

3924 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Getlik et al.
172.49, 160.92, 157.82, 153.61, 152.38, 146.63, 139.11, 138.92,
137.74, 137.18, 135.86, 133.95, 129.34, 128.46, 128.12, 127.19,
125.14, 123.58, 121.59, 118.40, 115.73, 112.26, 96.12, 32.40, 30.60,
29.71, 21.35, 10.01. HRMS (ESI-MS) calcd: 563.287 75 for
C32H35N8O2 [M + H+]. Found: 563.287 26.
solvent B until 90 min. The mass spectrometer (Thermo LTQ) was
equipped with a standard electrospray ion source (source voltage
of 4 kV). An automatic MS/MS analysis was performed for the
most intense peaks (minimal signal intensity of 10 000 required)
in a triple play experiment (normal MS, zoom scan of the most
intense peaks, followed by MS/MS in the case where the charge
state was 2 or higher). A 35% normalized collision energy was
used for MS/MS analysis.
Kinase Expression and Purification. N-Terminal His tag
constructs of human p38R and chicken cSrc (residues 251-533)
containing a PreScission protease cleavage site were expressed and
purified from E. coli. The p38R construct was cloned into a pOPINE
vector and was transformed as an N-terminal His-tag construct with
Precision protease cleavage site into BL21(DE3) E. coli. Cultures
were grown at 37 °C until an OD600 of 0.6 was attained, cooled in
30 min to room temperature, and then induced with 1 mM IPTG
for overnight (∼20 h) expression at 18 °C while shaking at 160
rpm. Cells were lysed in buffer A (50 mM Tris, pH 8.0, 500 mM
NaCl + 5% glycerol + 25 mM imidazole) and loaded onto a 30
mL Ni column (self-packed), washed with 3 column volumes (CV)
of Ni buffer A and then eluted with a 0-50% linear gradient using
Ni buffer B (Ni buffer A + 500 mM imidazole) over 2 CV. The
protein was cleaved by incubating with PreScission protease (50
µg/mL final concentration) in a 12-30 mL capacity 10-MWCO
dialysis cassette (Thermo Scientific) overnight at 4 °C in dialysis
buffer (50 mM Tris, pH 7.5, 5% glycerol, 150 mM NaCl, 1 mM
EDTA, 1 mM DTT). The protein was then centrifuged for 15 min
at ∼13 000 rpm to remove any precipitate that may have formed
during the cleavage step. The supernatant was then taken and diluted
at least 4-fold in anion buffer A (50 mM Tris, pH 7.4, 5% glycerol,
50 mM NaCl, 1 mM DTT) and loaded onto a 1 mL Sepharose Q
FF column (GE Healthcare) and washed with 10 CV of anion buffer
A. The protein was eluted with a 0-100% linear gradient of anion
buffer B (anion buffer A + 600 mM NaCl) over 20 CV. The protein
was pooled and concentrated down to 2 mL and passed through a
Sephadex HiLoad 26/60 Superdex 75 column equilibrated with size
exclusion buffer (20 mM Tris, pH 7.4, 5% glycerol, 200 mM NaCl,
1 mM DTT) at a rate of 2 mL/min. The eluted protein was then
concentrated to ∼10 mg/mL, aliquoted, and frozen at -80 °C. The
chicken cSrc gene (residues 251-533) was codon-usage optimized
for bacterial expression and synthesized synthetically (Geneart AG,
Regensburg, Germany). The chicken cSrc gene was cloned into a
pOPINF vector to generate an N-terminal His tag construct
containing a PreScission protease cleavage site. The plasmid was
transformed into BL21(DE3) codon + RIL E. coli for expression.
Briefly, cultures shaking at 200 rpm were grown in TB media
(containing 1% w/v glucose, chloramphenicol, and ampicillin) until
an OD600 of ∼0.2 was attained. The cultures were then cooled to
20 °C for 1 h prior to induction with 0.3 mM IPTG. The expression
continued overnight (approximately 20 h) at 20 °C. The protein
was purified using protocols similar to those described previously,14,50
with the exception of using PreScission protease (50 µg/mL final
concentration) to cleave the N-terminal His tag. Following size
exclusion, the eluted protein was concentrated to ∼10 mg/mL in
size exclusion buffer (50 mM Tris, pH 8.0, 100 mM NaCl, 5% v/v
glycerol, 1 mM DTT), aliquoted, and frozen at -80 °C.
Determination of Kd, kon, koff. Fluorophore labeled kinases were
generated as previously described.20 Screening initiatives were
carried out for acrylodan-labeled cSrc in 384-well plates. Stocks
of candidate compounds were prepared in DMSO at 20× the final
desired concentration. Compounds were mixed with labeled cSrc
in triplicate at final concentrations of 10 and 50 µM. Each well
contained 1 µL of compound and 19 µL of measurement buffer
(+0.01% v/v Brij-35) containing 100 nM kinase (5% v/v DMSO
after mixing). Plates were covered with an adhesive aluminum foil
and incubated for 15-30 min at room temperature prior to
measurement of emission intensities at 445, 475, and 505 nm using
a Tecan Safire2 plate reader. Acrylodan was excited at 386 nm.
Binding was measured using a ratio of λ445/λ475 while inhibitor
binding mode (DFG-in or DFG-out) was revealed by the ratio of
λ505/λ475. Potential hits were subjected to further titration studies
in cuvettes or 96-well plates to obtain Kd values. Kinetic measure-
ments were made by placing labeled cSrc into cuvettes with a
rapidly stirring mini stir bar and monitoring changes in acrylodan
fluorescence upon addition of a single dose of each inhibitor.
Inhibitors were dissolved in 100% DMSO stocks and delivered to
the sample through the injection port above the sample chamber.
At equilibrium, a 10-fold excess of unlabeled kinase was added to
extract the bound inhibitor from the labeled kinase. The resulting
fluorescence changes from binding and dissociation of the ligand
were well fitted to a first-order decay function to determine the
observed rate constants for dissociation (koff) as well as binding
(kon) for a single dose of each compound.
Crystallization and Structure Determination of cSrc-dasat-
inib, cSrc-3b and cSrc-T338M-3b. For the cSrc-dasatinib, cSrc-
3b and cSrc-T338M-3b complex structures, 500 µM inhibitor
(prepared in DMSO) was pre-incubated along with 180 µM wild
type cSrc or cSrc-T338M (stored in 20 mM Tris pH 8.0, 100 mM
NaCl, 1 mM DTT) for 4 hr to form the enzyme-inhibitor complex
prior to crystallization. In the case of dasatinib, crystals were grown
using the hanging drop method at (20 °C) after mixing 1 µL
protein-inhibitor solution with 0.5 µL reservoir solution (122 mM
MES (pH 6.4), 11% PEG 20000, 22.5% (v/v) glycerol). In the case
of 3b, crystals were grown using the sitting drop method at (20
°C) after mixing 0.2 µL protein-inhibitor complex and 0.2 µL
reservoir solution (85 mM MES (pH 6.5), 10.2% PEG 20000, 15%
(v/v) glycerol). Drops were pipetted using a Mosquito Nanodrop
crystallization robot (TTP LabTech Ltd., Melbourn, UK). All
crystals were directly frozen without the addition of glycerol.
Diffraction data of all cSrc-inhibitor complex crystals were
collected at the PX10SA beamline of the Swiss Light Source (PSI,
Villingen, Switzerland) to a resolution of 2.2 Å for cSrc-dasatinib
and 2.6 Å for cSrc-3b and cSrc-T338M-3b, using wavelengths close
to 1 Å. All data sets were processed with XDS51 and scaled using
XSCALE.51
Analysis of cSrc Labeling by HPLC and Mass Spectrometry.
Proteins were trypsinized according to standard procedures prior
to HPLC and mass spectrometry analysis to confirm the conjugation
of the fluorophore to the desired protein fragment. Unlabeled and
labeled cSrc (60 µg) samples were incubated separately with
proteomics grade trypsin (3 µg) in 55 mM NH4CO3 with 10% v/v
acetonitrile. Samples were incubated overnight at 37 °C, frozen in
liquid nitrogen, and lyophilized. The lyophilized powder was then
resuspended in 75 µL of water for analysis. Digested peptide
fragments were then separated and purified using an HPLC (Agilent
1100 series) equipped with a binary pump, thermostated autosam-
pler, and diode array detector. Samples were passed through a
Waters (Milford, MA) Atlantis dC18 column (2.1 mm × 150 mm)
with 3 µm particle size at ambient temperature. Samples were run
at 0.2 mL/min with the following gradient: 100% solvent A (0.1%
formic acid in water) for 5 min, ramping up to 60% solvent B (0.1%
formic in acetonitrile) with a linear gradient in 55 min, then
increasing to 80% solvent B in 10 min before holding at 80%
Structure Determination and Refinement of cSrc-Dasat-
inib, cSrc-3b, and cSrc-T338M-3b. All three cSrc-inhibitor
complex structures were solved by molecular replacement with
PHASER52 using the published cSrc structure 2OIQ53 as template.
The two cSrc molecules in the asymmetric unit were manually
modified using the program COOT.54 The model was first refined
with CNS55 using simulated annealing to remove model bias. The
final refinement was performed with REFMAC5.56 Inhibitor
topology files where generated using the Dundee PRODRG2
server.57 Refined structures were validated with PROCHECK.58
Detailed data, refinement, and Ramachandran statistics are in
Supporting Information Table 1. PyMOL59 was used to produce
the figures.

Hybrid Compound Design
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3925
Kinetics Assay for IC50 Determination. IC50 determinations for
cSrc kinases were measured with the HTRF KinEASETM-TK assay
from Cisbio according to the manufacturer’s instructions. A
biotinylated poly-Glu-Tyr substrate peptide was phosphorylated by
cSrc. After completion of the reaction, an anti-phosphotyrosine
antibody labeled with europium cryptate and streptavidin labeled
with the fluorophore XL665 were added. The FRET between
europium cryptate and XL665 was measured to quantify the
phosphorylation of the substrate peptide. ATP concentrations were
set at their respective Km values (15 µM for the wild type cSrc and
1 µM for cSrc-T338M), and 100 nM substrate was used for both
wild type and drug resistant cSrc. Kinase, substrate peptide, and
inhibitor were preincubated for 2 h before the reaction was started
by addition of ATP. A Tecan Safire2 plate reader was used to
measure the fluorescence of the samples at 620 nm (Eu-labeled
antibody) and 665 nm (XL665 labeled streptavidin) 60 µs after
excitation at 317 nm. The quotient of both intensities for reactions
made with eight different inhibitor concentrations was fit to a Hill
four-parameter equation to determine IC50 values. Each reaction
was performed in duplicate, and at least three independent
determinations of each IC50 were made.
Cell Culture. PC3 and DU145 were generously provided by
Dr. Roman Thomas (Max Planck Institute for Neurological
Research, Cologne, Germany). The cells were cultured in Dulbec-
co’s modified Eagle’s medium (DMEM) supplemented with 10%
heat-inactivated fetal bovine serum (FBS) and 100 units/mL
penicillin per streptomycin. Cells were cultured at 37 °C in
humidified air containing 5% CO2. After inhibitor treatment (5 h),
the cells were washed twice in cold phosphate-buffered saline (PBS)
and then lysed for 10 min on ice in lysis buffer (20 mM Tris-HCl,
pH 7.5, 150 mM NaCl, 1% Triton, 1 mM Na2EDTA, 1 mM EGTA,
2.5 mM sodium pyrophosphate, 1 mM ꢀ-glycerophosphate, 1 mM
Na3VO4, 1 µg/mL leupeptin, 1 mM PMSF, and common protease
inhibitors). Subsequently, cells were centrifuged for 20 min at
20000g and at 4 °C. The supernatant was subjected to immunoblot
analysis.
compound purities, and crystal X-ray structure data for 3b and
dasatinib. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) Gschwind, A.; Fischer, O. M.; Ullrich, A. The discovery of receptor
tyrosine kinases: targets for cancer therapy. Nat. ReV. Cancer 2004,
4, 361–370.
(2) Backes, A. C.; Zech, B.; Felber, B.; Klebl, B.; Mu¨ller, G. Small-
molecule inhibitors binding to protein kinases. Part I: exceptions from
the traditional pharmacophore approach of type I inhibition. Expert
Opin. Drug DiscoVery 2008, 3, 1409–1425.
(3) Backes, A. C.; Zech, B.; Felber, B.; Klebl, B.; Mu¨ller, G. Small-
molecule inhibitors binding to protein kinase. Part II: the novel
pharmacophore approach of type II and type III inhibition. Expert Opin.
Drug DiscoVery 2008, 3, 1427–1449.
(4) Zhang, J.; Yang, P. L.; Gray, N. S. Targeting cancer with small
molecule kinase inhibitors. Nat. ReV. Cancer 2009, 9, 28–39.
(5) Bikker, J. A.; Brooijmans, N.; Wissner, A.; Mansour, T. S. Kinase
domain mutations in cancer: implications for small molecule drug
design. J. Med. Chem. 2009, 52, 1493–1509.
(6) Liu, Y.; Gray, N. S. Rational design of inhibitors that bind to inactive
kinase conformations. Nat. Chem. Biol. 2006, 2, 358–364.
(7) Apsel, B.; Blair, J. A.; Gonzalez, B.; Nazif, T. M.; Feldman, M. E.;
Aizenstein, B.; Hoffman, R.; Williams, R. L.; Shokat, K. M.; Knight,
Z. A. Targeted polypharmacology: discovery of dual inhibitors of
tyrosine and phosphoinositide kinases. Nat. Chem. Biol. 2008, 4, 691–
699.
(8) Du, J.; Bernasconi, P.; Clauser, K. R.; Mani, D. R.; Finn, S. P.;
Beroukhim, R.; Burns, M.; Julian, B.; Peng, X. P.; Hieronymus, H.;
Maglathlin, R. L.; Lewis, T. A.; Liau, L. M.; Nghiemphu, P.;
Mellinghoff, I. K.; Louis, D. N.; Loda, M.; Carr, S. A.; Kung, A. L.;
Golub, T. R. Bead-based profiling of tyrosine kinase phosphorylation
identifies SRC as a potential target for glioblastoma therapy. Nat.
Biotechnol. 2009, 27, 77–83.
(9) Schenone, S.; Zanoli, S.; Brullo, C.; Crespan, E.; Maga, G. Current
advances in the development of anticancer drugs targeting tyrosine
kinases of the Src family. Curr. Drug Ther. 2008, 3, 158–176.
(10) Chang, Y. M.; Bai, L.; Liu, S.; Yang, J. C.; Kung, H. J.; Evans, C. P.
Src family kinase oncogenic potential and pathways in prostate cancer
as revealed by AZD0530. Oncogene 2008, 27, 6365–6375.
(11) Yeatman, T. J. A renaissance for SRC. Nat ReV Cancer. 2004, 4, 470–
480.
Immunoblot Analysis of Src and FAK. Protein concentration
was measured using a spectrophotometer (ND-1000, peQLab).
Equal amounts of protein were separated by SDS-PAGE and
transferred to nitrocellulose membranes. Blots were blocked for
1 h in Tris-buffered saline with Tween-20 (TBST) supplemented
with 5% nonfat milk and subsequently incubated overnight at 4 °C
in primary antibody, namely, anti-phospho-FAK, anti-phospho-Src,
anti-FAK, and anti-Src. All antibodies were obtained from Cell
Signaling Technology. After being washed, blots were incubated
with secondary antibodies and then detected on film using the
enhanced chemiluminescence (ECL) detection system.
(12) Daub, H.; Specht, K.; Ullrich, A. Strategies to overcome resistance to
targeted protein kinase inhibitors. Nat. ReV. Drug DiscoVery 2004, 3,
1001–1010.
(13) Blencke, S.; Ullrich, A.; Daub, H. Mutation of threonine 766 in the
epidermal growth factor receptor reveals a hotspot for resistance
formation against selective tyrosine kinase inhibitors. J. Biol. Chem.
2003, 278, 15435–15440.
(14) Michalczyk, A.; Klu¨ter, S.; Rode, H. B.; Simard, J. R.; Gru¨tter, C.;
Rabiller, M.; Rauh, D. Structural insights into how irreversible
inhibitors can overcome drug resistance in EGFR. Bioorg. Med. Chem.
2008, 16, 3482–3488.
(15) Carter, T. A.; Wodicka, L. M.; Shah, N. P.; Velasco, A. M.; Fabian,
M. A.; Treiber, D. K.; Milanov, Z. V.; Atteridge, C. E.; Biggs, W. H.,
3rd; Edeen, P. T.; Floyd, M.; Ford, J. M.; Grotzfeld, R. M.; Herrgard,
S.; Insko, D. E.; Mehta, S. A.; Patel, H. K.; Pao, W.; Sawyers, C. L.;
Varmus, H.; Zarrinkar, P. P.; Lockhart, D. J. Inhibition of drug-resistant
mutants of ABL, KIT, and EGF receptor kinases. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 11011–11016.
(16) Azam, M.; Seeliger, M. A.; Gray, N. S.; Kuriyan, J.; Daley, G. Q.
Activation of tyrosine kinases by mutation of the gatekeeper threonine.
Nat. Struct. Mol. Biol. 2008, 15, 1109–1118.
(17) Yun, C. H.; Mengwasser, K. E.; Toms, A. V.; Woo, M. S.; Greulich,
H.; Wong, K. K.; Meyerson, M.; Eck, M. J. The T790M mutation in
EGFR kinase causes drug resistance by increasing the affinity for ATP.
Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2070–2075.
(18) Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.; Atteridge,
C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.; Edeen,
P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D. J.; Milanov,
Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.;
Wodicka, L. M.; Zarrinkar, P. P. A quantitative analysis of kinase
inhibitor selectivity. Nat. Biotechnol. 2008, 26, 127–132.
(19) Young, M. A.; Shah, N. P.; Chao, L. H.; Seeliger, M.; Milanov, Z. V.;
Biggs, W. H., 3rd; Treiber, D. K.; Patel, H. K.; Zarrinkar, P. P.;
Lockhart, D. J.; Sawyers, C. L.; Kuriyan, J. Structure of the kinase
domain of an imatinib-resistant Abl mutant in complex with the Aurora
kinase inhibitor VX-680. Cancer Res. 2006, 66, 1007–1014.
(20) (a) Simard, J. R.; Klu¨ter, S.; Gru¨tter, C.; Getlik, M.; Rabiller, M.;
Rode, H. B.; Rauh, D. A new screening assay for allosteric inhibitors
of cSrc. Nat. Chem. Biol., doi: 10.1038/nchembio.162. (b) Simard,
Acknowledgment. We thank Michael Weyand, Eckhard
Hofmann, Ingrid Vetter, Wulf Blanckenfeldt, and beamline
scientists at X10SA for expert assistance during data collection.
We thank the Dortmund Protein Facility for cloning, expressing,
and purifying some of the chicken cSrc and human p38R used
in these studies. We thank Roman Thomas (Max Planck Institute
for Neurological Research, Cologne, Germany) for the PC3 and
DU145 cancer cell lines and Lars Ruddigkeit for synthetic
intermediates. J.R.S. was funded by the Alexander von Hum-
boldt Foundation. Schering Plough, Bayer-Schering Pharma,
Merck-Serono, and BayerCrop Science are thanked for financial
support. The work was supported by the German Federal
Ministry for Education and Research through the German
National Genome Research Network-Plus (NGFN-Plus) (Grant
No. BMBF 01GS08102).
Supporting Information Available: The crystal structure of
dasatinib in complex with cSrc, synthesis scheme of pyrazoloureas,
1b modeled into the binding site of drug resistant cSrc-T338M,
1,3-meta and 1,4-para hybrid compounds modeled to inactive
human p38R, kinase selectivity profiles for 3b and 3e, rate constants
for the dissociation of 3a-e from acrylodan-labeled cSrc, table of

3926 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Getlik et al.
J. R.; Getlik, M.; Gru¨tter, C.; Pawar, V.; Wulfert, S.; Rabiller, M.;
Rauh, D.; Development of a fluorescent-tagged kinase assay system
for the detection and characterization of allosteric kinase inhibitors.
JACS 2009, in press.
(39) Owens, L. V.; Xu, L.; Craven, R. J.; Dent, G. A.; Weiner, T. M.;
Kornberg, L.; Liu, E. T.; Cance, W. G. Overexpression of the focal
adhesion kinase (p125FAK) in invasive human tumors. Cancer Res.
1995, 55, 2752–2755.
(40) Shor, A. C.; Keschman, E. A.; Lee, F. Y.; Muro-Cacho, C.; Letson,
G. D.; Trent, J. C.; Pledger, W. J.; Jove, R. Dasatinib inhibits migration
and invasion in diverse human sarcoma cell lines and induces apoptosis
in bone sarcoma cells dependent on SRC kinase for survival. Cancer
Res. 2007, 67, 2800–2808.
(41) Dar, A. C.; Lopez, M. S.; Shokat, K. M. Small molecule recognition
of c-Src via the imatinib-binding conformation. Chem. Biol. 2008, 15,
1015–1022.
(42) Copeland, R. A.; Pompliano, D. L.; Meek, T. D. Drug-target residence
time and its implications for lead optimization. Nat. ReV. Drug
DiscoVery 2006, 5, 730–739.
(43) Bradeen, H. A.; Eide, C. A.; O’Hare, T.; Johnson, K. J.; Willis, S. G.;
Lee, F. Y.; Druker, B. J.; Deininger, M. W. Comparison of imatinib
mesylate, dasatinib (BMS-354825), and nilotinib (AMN107) in an
N-ethyl-N-nitrosourea (ENU)-based mutagenesis screen: high efficacy
of drug combinations. Blood 2006, 108, 2332–2338.
(44) von Bubnoff, N.; Veach, D. R.; van der Kuip, H.; Aulitzky, W. E.;
Sanger, J.; Seipel, P.; Bornmann, W. G.; Peschel, C.; Clarkson, B.;
Duyster, J. A cell-based screen for resistance of Bcr-Abl-positive
leukemia identifies the mutation pattern for PD166326, an alternative
Abl kinase inhibitor. Blood 2005, 105, 1652–1659.
(45) Zunder, E. R.; Knight, Z. A.; Houseman, B. T.; Apsel, B.; Shokat,
K. M. Discovery of drug-resistant and drug-sensitizing mutations in
the oncogenic PI3K isoform p110 alpha. Cancer Cell. 2008, 14, 180–
192.
(21) Dumas, J.; Hatoum-Mokdad, H.; Sibley, R.; Riedl, B.; Scott, W. J.;
Monahan, M. K.; Lowinger, T. B.; Brennan, C.; Natero, R.; Turner,
T.; Johnson, J. S.; Schoenleber, R.; Bhargava, A.; Wilhelm, S. M.;
Housley, T. J.; Ranges, G. E.; Shrikhande, A. 1-Phenyl-5-pyrazolyl
ureas: potent and selective p38 kinase inhibitors. Bioorg. Med. Chem.
Lett. 2000, 10, 2051–2054.
(22) Pargellis, C.; Tong, L.; Churchill, L.; Cirillo, P. F.; Gilmore, T.;
Graham, A. G.; Grob, P. M.; Hickey, E. R.; Moss, N.; Pav, S.; Regan,
J. Inhibition of p38 MAP kinase by utilizing a novel allosteric binding
site. Nat. Struct. Biol. 2002, 9, 268–272.
(23) Sharma, S. V.; Bell, D. W.; Settleman, J.; Haber, D. A. Epidermal
growth factor receptor mutations in lung cancer. Nat. ReV. Cancer
2007, 7, 169–181.
(24) Hoelzemann, G.; Crassier, H.; Rautenberg, W. Pyrazole Derivatives
Having Tyrosinkinase Activity. WO2007017083 (A1), 2007.
(25) Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Discovering
high-affinity ligands for proteins: SAR by NMR. Science 1996, 274,
1531–1534.
(26) Nienaber, V. L.; Richardson, P. L.; Klighofer, V.; Bouska, J. J.;
Giranda, V. L.; Greer, J. Discovering novel ligands for macromolecules
using X-ray crystallographic screening. Nat. Biotechnol. 2000, 18,
1105–1108.
(27) Gill, A. L.; Frederickson, M.; Cleasby, A.; Woodhead, S. J.; Carr,
M. G.; Woodhead, A. J.; Walker, M. T.; Congreve, M. S.; Devine,
L. A.; Tisi, D.; O’Reilly, M.; Seavers, L. C.; Davis, D. J.; Curry, J.;
Anthony, R.; Padova, A.; Murray, C. W.; Carr, R. A.; Jhoti, H.
Identification of novel p38alpha MAP kinase inhibitors using fragment-
based lead generation. J. Med. Chem. 2005, 48, 414–426.
(28) Jacobs, M. D.; Caron, P. R.; Hare, B. J. Classifying protein kinase
structures guides use of ligand-selectivity profiles to predict inactive
conformations: structure of lck/imatinib complex. Proteins 2008, 70,
1451–1460.
(46) Crespo, A.; Zhang, X.; Fernandez, A. Redesigning kinase inhibitors
to enhance specificity. J. Med. Chem. 2008, 51, 4890–4898.
(47) Regan, J.; Breitfelder, S.; Cirillo, P.; Gilmore, T.; Graham, A. G.;
Hickey, E.; Klaus, B.; Madwed, J.; Moriak, M.; Moss, N.; Pargellis,
C.; Pav, S.; Proto, A.; Swinamer, A.; Tong, L.; Torcellini, C. Pyrazole
urea-based inhibitors of p38 MAP kinase: from lead compound to
clinical candidate. J. Med. Chem. 2002, 45, 2994–3008.
(48) Bagley, M. C.; Davis, T.; Dix, M. C.; Widdowson, C. S.; Kipling, D.
Microwave-assisted synthesis of N-pyrazole ureas and the p38alpha
inhibitor BIRB 796 for study into accelerated cell ageing. Org. Biomol.
Chem. 2006, 4, 4158–4164.
(29) Okram, B.; Nagle, A.; Adrian, F. J.; Lee, C.; Ren, P.; Wang, X.; Sim,
T.; Xie, Y.; Xia, G.; Spraggon, G.; Warmuth, M.; Liu, Y.; Gray, N. S.
A general strategy for creating “inactive-conformation” abl inhibitors.
Chem. Biol. 2006, 13, 779–786.
(30) Warner, S. L.; Bashyam, S.; Vankayalapati, H.; Bearss, D. J.; Han,
H.; Mahadevan, D.; Von, H. D. D.; Hurley, L. H. Identification of a
lead small-molecule inhibitor of the Aurora kinases using a structure-
assisted, fragment-based approach. Mol. Cancer Ther. 2006, 5, 1764–
1773.
(31) Shewchuk, L.; Hassell, A.; Wisely, B.; Rocque, W.; Holmes, W.; Veal,
J.; Kuyper, L. F. Binding mode of the 4-anilinoquinazoline class of
protein kinase inhibitor: X-ray crystallographic studies of 4-anilino-
quinazolines bound to cyclin-dependent kinase 2 and p38 kinase.
J. Med. Chem. 2000, 43, 133–138.
(32) Heron, N. M.; Anderson, M.; Blowers, D. P.; Breed, J.; Eden, J. M.;
Green, S.; Hill, G. B.; Johnson, T.; Jung, F. H.; McMiken, H. H.;
Mortlock, A. A.; Pannifer, A. D.; Pauptit, R. A.; Pink, J.; Roberts,
N. J.; Rowsell, S. SAR and inhibitor complex structure determination
of a novel class of potent and specific Aurora kinase inhibitors. Bioorg.
Med. Chem. Lett. 2006, 16, 1320–1323.
(33) Blair, J. A.; Rauh, D.; Kung, C.; Yun, C. H.; Fan, Q. W.; Rode, H.;
Zhang, C.; Eck, M. J.; Weiss, W. A.; Shokat, K. M. Structure-guided
development of affinity probes for tyrosine kinases using chemical
genetics. Nat. Chem. Biol. 2007, 3, 229–238.
(34) Stamos, J.; Sliwkowski, M. X.; Eigenbrot, C. Structure of the epidermal
growth factor receptor kinase domain alone and in complex with a
4-anilinoquinazoline inhibitor. J. Biol. Chem. 2002, 277, 46265–46272.
(35) Hunter, C. A.; Singh, J.; Thornton, J. M. Pi-pi interactions: the
geometry and energetics of phenylalanine-phenylalanine interactions
in proteins. J. Mol. Biol. 1991, 218, 837–846.
(36) Schaller, M. D. Biochemical signals and biological responses elicited
by the focal adhesion kinase. Biochim. Biophys. Acta 2001, 1540, 1–
21.
(37) Calalb, M. B.; Polte, T. R.; Hanks, S. K. Tyrosine phosphorylation of
focal adhesion kinase at sites in the catalytic domain regulates kinase
activity: a role for Src family kinases. Mol. Cell. Biol. 1995, 15, 954–
963.
(38) Nowakowski, J.; Cronin, C. N.; McRee, D. E.; Knuth, M. W.; Nelson,
C. G.; Pavletich, N. P.; Rogers, J.; Sang, B. C.; Scheibe, D. N.;
Swanson, R. V.; Thompson, D. A. Structures of the cancer-related
Aurora-A, FAK, and EphA2 protein kinases from nanovolume
crystallography. Structure 2002, 10, 1659–1667.
(49) Zhang, L. H.; Zhu, L. Novel Process for Synthesis of Heteroaryl-
Substituted Urea Compounds. WO/2001/004115, 2001.
(50) Seeliger, M. A.; Young, M.; Henderson, M. N.; Pellicena, P.; King,
D. S.; Falick, A. M.; Kuriyan, J. High yield bacterial expression of
active c-Abl and c-Src tyrosine kinases. Protein Sci. 2005, 14, 3135–
3139.
(51) Kabsch, W. Automatic processing of rotation diffraction data from
crystals of initially unknown symmetry and cell constants. J. Appl.
Crystallogr. 1993, 26, 795–800.
(52) Read, R. J. Pushing the boundaries of molecular replacement with
maximum likelihood. Acta Crystallogr., Sect. D: Biol. Crystallogr.
2001, 57, 1373–1382.
(53) Seeliger, M. A.; Nagar, B.; Frank, F.; Cao, X.; Henderson, M. N.;
Kuriyan, J. c-Src binds to the cancer drug imatinib with an inactive
Abl/c-Kit conformation and a distributed thermodynamic penalty.
Structure 2007, 15, 299–311.
(54) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular
graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126–
2132.
(55) Bru¨nger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.;
Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L.
Crystallography & NMR system: a new software suite for macromo-
lecular structure determination. Acta Crystallogr., Sect. D: Biol.
Crystallogr. 1998, 54, 905–921.
(56) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of
macromolecular structures by the maximum-likelihood method. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240–255.
(57) Schu¨ttelkopf, A. W.; van Aalten, D. M. PRODRG: a tool for high-
throughput crystallography of protein-ligand complexes. Acta Crys-
tallogr., Sect. D: Biol. Crystallogr. 2004, 60, 1355–1363.
(58) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M.
PROCHECK: a program to check the stereochemical quality of protein
structures. J. Appl. Crystallogr. 1993, 26, 283–291.
(59) DeLano, W. L. The PyMOL Molecular Graphics System; DeLano
Scientific LLC: Palo Alto, CA, 2002; http://www.pymol.org.
JM9002928