Targeting gain of function and resistance mutations in Abl and KIT by hybrid compound design

Article abstract of DOI:10.1021/jm4004076

Mutations in the catalytic domain at the gatekeeper position represent the most prominent drug-resistant variants of kinases and significantly impair the efficacy of targeted cancer therapies. Understanding the mechanisms of drug resistance at the molecular and atomic levels will aid in the design and development of inhibitors that have the potential to overcome these resistance mutations. Herein, by introducing adaptive elements into the inhibitor core structure, we undertake the structure-based development of type II hybrid inhibitors to overcome gatekeeper drug-resistant mutations in cSrc-T338M, as well as clinically relevant tyrosine kinase KIT-T670I and Abl-T315I variants, as essential targets in gastrointestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML). Using protein X-ray crystallography, we confirm the anticipated binding mode in cSrc, which proved to be essential for overcoming the respective resistances. More importantly, the novel compounds effectively inhibit clinically relevant gatekeeper mutants of KIT and Abl in biochemical and cellular studies.

Full text of DOI:10.1021/jm4004076

Article
pubs.acs.org/jmc
Targeting Gain of Function and Resistance Mutations in Abl and KIT
by Hybrid Compound Design
Andre Richters,† Julia Ketzer,‡ Matthaus Getlik,† Christian Grutter,† Ralf Schneider,§
́
̈
̈
Johannes M. Heuckmann,⊥ Stefanie Heynck,⊥ Martin L. Sos,⊥ Anu Gupta,▽ Anke Unger,#
Carsten Schultz-Fademrecht,# Roman K. Thomas,⊥,∥ Sebastian Bauer,‡,* and Daniel Rauh†,§,*
†Department of Chemistry and Chemical Biology, Technical University of Dortmund, Otto-Hahn-Strasse 6, D-44227 Dortmund,
Germany
‡Department of Medical Oncology, Sarcoma Center, West German Cancer Center, University Duisburg-Essen Medical School,
Hufelandstrasse 55, D-45122 Essen, Germany
§Chemical Genomics Centre of the Max-Planck-Society, Otto-Hahn-Strasse 15, D-44227 Dortmund, Germany
⊥Department of Translational Genomics, University of Cologne, Weyertal 115b, D-50931 Cologne, Germany
∥Department of Pathology, University of Cologne, Joseph-Stelzmann Strasse 9, D-50931 Cologne, Germany
▽Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, United States
#Lead Discovery Center GmbH, Otto-Hahn-Strasse 15, D-44227 Dortmund, Germany
S
* Supporting Information
ABSTRACT: Mutations in the catalytic domain at the
gatekeeper position represent the most prominent drug-
resistant variants of kinases and significantly impair the efficacy
of targeted cancer therapies. Understanding the mechanisms of
drug resistance at the molecular and atomic levels will aid in
the design and development of inhibitors that have the
potential to overcome these resistance mutations. Herein, by
introducing adaptive elements into the inhibitor core structure,
we undertake the structure-based development of type II hybrid
inhibitors to overcome gatekeeper drug-resistant mutations in
cSrc-T338M, as well as clinically relevant tyrosine kinase KIT-
T670I and Abl-T315I variants, as essential targets in gastro-
intestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML). Using protein X-ray crystallography, we confirm the
anticipated binding mode in cSrc, which proved to be essential for overcoming the respective resistances. More importantly, the novel
compounds effectively inhibit clinically relevant gatekeeper mutants of KIT and Abl in biochemical and cellular studies.
sequencing efforts of primary tumors show a high incidence of
mutations in the active site of targeted kinases, which are responsible
for this resistance.14 Thus, drug resistance is a major problem in the
long-term treatment with targeted cancer therapies.15−17 Treatment
of CML patients with imatinib (Figure 1a), for instance, leads on
average to the incidence of drug resistance mutations in the kinase
domain of Bcr-Abl or intolerance to imatinib in 30% of examined
patient populations in the first five years.13 Even more dramatically,
in the case of patients suffering from solid GISTs, 14% of patients
initially do not respond to imatinib treatment and 50% will develop
resistance mutations in the kinase domain of the stem cell growth
factor receptor KIT within the first two years.9 Likewise, the efficacy
of gefitinib in the treatment of NSCLC is significantly impaired
by acquired resistance mutation T790M at the gatekeeper position
of EGFR and renders even second-generation inhibitors that
INTRODUCTION
■
Protein kinases represent a major enzyme family comprised of
more than 500 members encoded in the human genome, which
are involved in several signal transduction processes, including
those responsible for cell proliferation, apoptosis, and cell
survival.1−3 Since deregulation of signaling pathways, especially
by constitutive activation of kinases, often leads to the onset of
various diseases, including cancer, the inhibition of kinases has
been at the leading edge of cancer research for decades.4
Various kinase inhibitors bind to the ATP binding site (type I
and II inhibitors)5−8 and have proven to be powerful tools in
the treatment of cancer. Imatinib targets tyrosine kinases such
as KIT in gastrointestinal stromal tumors (GISTs)9,10 and Bcr-Abl
in chronic myelogenous leukemia (CML).11 Similarly, gefitinib
targets EGFR in non-small-cell lung cancer (NSCLC).12 Although
these inhibitors are very successful in early disease stages in selected
patient populations, the emergence of drug resistance during later
stages of treatment is becoming an ever-increasing challenge.13 DNA
Received: March 19, 2013
© XXXX American Chemical Society
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Figure 1. (A) General scaffold of a 1,4-fused type II hybrid inhibitor, 1, composed of type I and type III fragments, as well as hybrid compound 2, as
an initial rationally designed molecule to target cSrc wild-type and T338M. Imatinib and ponatinib represent approved type II inhibitors for the
treatment of GISTs and CML. (B) Schematic overview on how to circumvent steric repulsion with bulky gatekeepers (e.g., methionine) in drug-
resistant mutant kinases.
irreversibly modify a rare cysteine at the lip of the catalytic cleft with
only limited clinical impact.18,19 This demonstrates that the
development of new and selective inhibitors that can inhibit clinically
relevant kinases is still of major importance for the treatment of
diseases such as GIST, CML, and NSCLC.
One of the most prominent and striking mutations is the
replacement of the gatekeeper residue, a position that is known
to control access to the ATP-binding site affecting inhibitor
selectivity, with a bulkier and often more lipophilic residue, as is
the case for T670I of the KIT oncogenic drug-resistant variant
in GIST. The primary gain of function mutation V559D in the
juxtamembrane region of KIT leads to constitutive activation,
while the secondary mutation T670I at the gatekeeper position
is accompanied by insensitivity toward imatinib as a first line
therapy.20,21 Similarly the oncogenic fusion protein Bcr-Abl in
CML results in stabilization of the active kinase conformation
leading to a constitutively active protein. Additionally, the
amino acid exchange of threonine for isoleucine at the
gatekeeper position 315 (T315I) causes a steric repulsion
that prohibits inhibitors from binding within the active site and
causes resistance to imatinib.22 Moreover, this substitution at
position 315 represents the only variant out of more than
B
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Table 1. Overview of Rationally Designed Small Molecule Library Consisting of Variably Fused 1,4- and 1,3-Fused Hybrid
Derivatives as Well as Fragments and Compounds with Replaced Pyrazolo Part
50 drug-resistant mutations occurring in the catalytic domain of
Bcr-Abl that is presently unfeasible to address in cancer therapy
with small molecule inhibitors23 with one exception: the type II
inhibitor ponatinib (Figure 1a) recently received accelerated
approval by the FDA while still running in phase III clinical
trials, but treatment potentially produces severe side effects
such as hepatotoxicity and arterial thrombosis.24,25 Therefore,
current efforts in kinase research focus on overcoming these
mutant variants by developing allosteric inhibitors that
exclusively bind outside the ATP binding site and lock the
kinase in its inactive form.26−29
Recently, we presented the structure-guided development of
type II inhibitors of cSrc. Fusion of a pyrazolo urea scaffold (type
III, “allosteric”) with a quinazoline core (type I, ATP-competitive)
resulted in potent type II cSrc inhibitors (1, Figure 1a) that lock
the kinase in the inactive DFG-out conformation; these inhibitors
were also active on the drug-resistant T338M gatekeeper mutant
of cSrc. Protein X-ray crystallography revealed the rotational
flexibility of a central 1,4-substituted phenyl ring of the inhibitor,
which allows the hybrids to circumvent sterically demanding
gatekeepers.30 Here, we present the structure-guided optimization
of those inhibitors to target the clinically relevant kinases KIT and
Abl and their respective gatekeeper mutant forms T670I and
T315I in biochemical and cellular systems.
RESULTS
■
Structure-Based Design of Type II Hybrid Inhibitors
against cSrc, KIT, and Abl. Since cSrc, KIT, and Abl belong
to closely related families of tyrosine kinases, we used the
gatekeeper mutant cSrc T338M as an initial model system to
transfer to KIT T670I and Abl T315I. To evaluate this, we
generated a superimposed model of both (Figure S1,
Supporting Information), based on the X-ray structure of
cSrc T338M in complex with 2a (PDB 3F3W).30 From this
model, we predicted that a binding mode highly similar to cSrc
would occur, suggesting that the new hybrid compounds would
also show an inhibitory activity for KIT and Abl, as well as their
gatekeeper mutant forms. The quinazoline N1 addresses the
hinge, and the urea moiety is sandwiched between helix C and
the DFG-motif of the kinase, which adopts its “out”
conformation. A phenyl ring allowing for rotational flexibility
links these parts of the inhibitor and interacts with the DFG-
phenylalanine through a favorable edge-to-face orientation.31
The pyrazolo part points into the allosteric pocket, which is
accessible only in the kinase’s inactive conformation.1 Taking
into account the hybrid compound’s ability to overcome
gatekeeper mutations as for cSrc T338M, we proposed that
these small molecules would be promising binders to inhibit the
gatekeeper mutated forms of KIT T670I and Abl T315I.
C
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a
Scheme 1. Synthesis of a Subset of Quinazoline Derivatives and Fragments
a
Reagents and conditions: (i) Pivaloyl acetonitrile, conc. HCl, EtOH, 90 °C; (ii) Troc-Cl, NaOH, EtOAc/H2O, 0 °C to rt; (iii) methylamine
hydrochloride, DIPEA, DMSO, rt; (iv) N-Boc phenylenediamine, DIPEA, DMSO, rt; (v) ammonium formate, Pd/C, EtOH, 90 °C; (vi) Fmoc-Cl,
NaHCO3, dioxane/H2O, rt; (vii) 4 M HCl in dioxane, rt; (viii) 4-chloro-6-aminoquinazoline, DIPEA, DCM, rt; (ix) iron chipping (activated with
1 N HCl), AcOH, EtOH, reflux; (x) corresponding acid chloride of f−i; THF, base (DIPEA or NMP), THF or DCM, rt; (xi) piperidine or
morpholine in DMF, rt. The framed intermediate molecule was further used to generate other hybrid derivatives in a different synthetic route (See
Supporting Information, Schemes S1 and S2).
Our optimization efforts were directed toward the extension
of the hybrids at the quinazoline’s 6-position with H-bond
acceptor moieties (e.g., 3g,h, Table 1). We speculated that
introduction of polar motifs would lead to the formation of
an additional contact at the lip of the active site to Asp348 in
cSrc (data not shown), as well as to the corresponding residues
Asn680 in KIT and Asp325 in Abl as shown in the case of 3g
(Figure S1, Supporting Information). In addition, the moderate
solubility of the original compound would be increased. Further-
more, we exchanged the meta-positioned methyl group of the
pyrazole connected phenyl ring with an amino moiety to
improve the interaction within a polar subgroove adjacent to
the allosteric binding site, which is formed by the polar side
chain residues Glu310 (helix C), Asp404 (activation loop), and
catalytic Lys295. We assumed that the ligand’s polar interplay
with this region would give further benefit to the affinity of these
hybrid compounds toward gatekeeper mutant forms of cSrc
(T338M), KIT (T670I), and Abl (T315I). We also replaced
the pyrazolo part by a 4-chloro-3-trifluormethylphenyl moiety
(6), the hydrophobic motif of the known multikinase inhibitor
sorafenib,32 to study structure−activity relationships (SARs)
among different implemented type III scaffolds and to achieve
further increases in potency (Table 1).
Synthesis of a Focused Library of Hybrid Compounds
and Fragments. We synthesized a focused library of variably
fused type II inhibitors (Table 1) and paid particular attention
to the derivatization of the quinazoline 6-position with various
polar moieties frequently utilized in kinase inhibitor research
(e.g., morpholine or methyl-piperazine).33,34 Since they
exhibited different structural compositions, we expected these
inhibitors to show a distinct SAR regarding their potency
toward the investigated kinases. We especially expected the
D
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Table 2. IC50 Determinations of Hybrid Compounds and Fragments on Different Target Kinases
IC50 (nM)
compd
3a
cSrcWT
cSrcT338M
AblWT
AblT315I
cKITWT
cKITV559D/T670I
34.2 22.6
19.9 11.5
12.0 0.5
37.9 11.8
145 47
1.2 0.5
1.1 0.6
1.1 0.7
4.7 2.1
39.3 6.1
304 94
a
126 82.0
157 76
42 23
561 222
355 116
81 23
120 43
3620 1180
441 124
1170 450
176 47
820 304
a
69.3 19.3
7.3 5.4
6.1 1.9
3.3 2.2
20.9 6.5
7.6 1.6
2.6 0.5
3.5 1.9
5.7 3.4
29.2 21.0
30.1 22.0
a
637 91
38.0 16.4
7.6 3.8
24.0 3.7
305 69
22.1 13.3
2.7 1.7
6.9 2.7
9.2 3.7
a
3b
33.0 5.1
3c
5.9 3.7
73 36
3d
33.3 10.2
40 20
3e
211 120
1273 301
112 45
124 54
61 53
3f
1.7 1.6
3g
1.3 0.2
3h
0.9 0.4
3i
3.5 1.5
379 70
4a
a
a
a
a
a
a
9
23
a
7
4
4b
a
a
5a
a
a
5b
a
a
a
a
a
6a
a
a
a
4.5 2.2
1.0 0.4
21.9 6.1
21.6 7.0
0.7 0.3
1.7 0.2
1.1 0.3
a
6b
3037 200
a
a
923 305
155 84
2b
14
1
23
4
388 88
19.9 2.7
1613 938
2.9 1.2
141 50
a
imatinib
sunitinib
ponatinib
dovitinib
2784 481
555 48
a
≥100000
134 41
156 27
50.1 15.4
504 102
1.0 0.5
228 67
264 92
4.9 0.6
48.3 5.2
0.9 0.3
80.8 35.1
450 65
a
No inhibition at 100 μM compound concentration.
1,4-fused hybrids to perturb gatekeeper mutated forms of the
kinases. The panel of inhibitors also included scaffolds with the
pyrazolo part removed (6a,b) to (a) study the relevance of this
structural motif concerning the binding affinity of the hybrid
compounds and (b) further improve the inhibitory activity.
Additionally, we incorporated type III pyrazolo-urea inhibitors
(5a,b) into the set of tested compounds.
3b, and 4a. Reduction with Pd/C (for 3a) or SnCl2 (for 3b, 4a)
to the corresponding amines 3c, 3d, and 4b was conducted to
complete this subset of derivatives.
Compounds 6a and 6b (Scheme S2) were made by initial
reaction of 1-chloro-4-isocyanato-2-(trifluoromethyl)benzene
with tert-butyl 4-aminophenylcarbamate. Boc-cleavage led to
15, which then was treated with 4-chloro-6-nitroquinazoline
to generate quinazoline urea 6a in a SNAr reaction. Finally,
reduction with SnCl2 was performed to prepare amine 6b.
Detailed synthetic procedures are described in the Experimental
Section and Supporting Information.
Hybrid compounds with different variations on the quinazo-
line 6-position were synthesized using a generic route (Scheme 1)
starting with a Pinner-like cyclization of m-nitrophenyl
hydrazine and pivaloyl acetonitrile to generate pyrazole 7.
The primary amine of 7 was then protected with 2,2,2-
trichloroethyl chloroformate (Troc), yielding carbamate 8,
which subsequently was reacted with various primary amines to
create the central urea moiety.30 Fragments 5a and 5b were
generated by treatment of 8 with methylamine hydrochloride to
build up 5a, and successive reduction with Pd/C provided
the corresponding amine 5b. Intermediates 9 and 10 were
synthesized by reacting single Boc-protected 1,3- and 1,4-
phenylenediamines with 8. The 1,4-fused precursor 9 was then
used to assemble quinazoline-pyrazoloureas 3e−i by initial
reduction to an intermediary amine and successive introduction
of the orthogonal Fmoc-protecting group, providing 11. An
orthogonal protecting group strategy was essential to ensure
selective derivatization of the quinazoline 6-position in 3e.
Next, 6-nitro-4-chloro-quinazoline was added via a two-step
process, starting with Boc-deprotection and subsequent coupling
of the quinazoline to form 12 by nucleophilic aromatic
substitution (SNAr). Reduction with Fe/AcOH provided 3e
which was further derivatized through direct amide formation with
(in situ prepared) acid chlorides. Finally, Fmoc-cleavage provided
compounds 3f−i.
Activity-Based in Vitro Characterization of Hybrid
Compounds against cSrc. We characterized the focused
compound collection against cSrc using an activity-based assay
that quantifies phosphorylation of an artificial substrate by a
particular kinase through homogeneous time-resolved FRET
measurements (see Experimental Section). The observed IC50
values (Table 2) confirmed an improved activity of the 1,4-
fused hybrid compounds 3f−h (∼1 nM) with respect to both
cSrc wild-type and its T338M mutant. In comparison to the
most potent first generation hybrid compound (2b), this
corresponded to a 14- and 23-fold increase in potency against
wild-type and mutant cSrc, respectively. Converting the
pyrazolo group into a 4-chloro-3-trifluormethylphenyl moiety
significantly decreased the affinity (6a,b) and demonstrated
that the pyrazolo part is an important structural feature for
preserving the binding affinity of the hybrids within the
allosteric site of cSrc (Table 2). However, this element on its
own (5a,b) did not show any inhibitory activity toward the
protein due to the absence of the ability to lock the kinase in its
inactive DFG-out conformation. Moreover, we illustrated a
clear SAR among different derivatives concerning their efficacy
against both cSrc variants. In general, the 1,4-fused hybrids
3f−h were equally potent against both the wild-type and mutant
kinase, while 1,3-fused analogs (4a,b) completely lost their
potency against the mutant cSrc carrying the bulky methionine
gatekeeper. This was to be expected from our previous study,
Quinazoline derivatives 3a, 3b, and 4a were prepared via
Boc-deprotection of intermediates 9 and 10 (see above). The
corresponding amines 13 and 14 (Scheme S1, Supporting
Information) were reacted with 6-nitro-4-chloroquinazoline or
4-chloroquinazoline, respectively, resulting in compounds 3a,
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Figure 2. 1,4-Substituted hybrid compounds in complex with wild-type cSrc. Diagrams of the experimental electron densities of cSrc−3h at 2.7 Å
(A) and cSrc-3g at 3.3 Å (B) resolution are shown (2Fo − Fc map contoured at 1σ). Hydrogen-bond interactions of the inhibitors with helix C
(turquoise), the DFG-motif (pink), and the hinge region (orange) are shown by red dotted lines. The kinase domain is in the inactive DFG-out
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 Met341, 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 Phe405 (DFG motif) in a favorable edge-to-face orientation. (C) The aromatic amine points into a polar subpocket
that is formed by three polar amino acid side chains (Lys295, Glu310, and Asp404) and forms polar interactions with a water molecule (red sphere).
A detailed illustration of this interaction, including electron density data, is provided in Figure S2, Supporting Information.
in which we showed that the rigid 1,3-hybrid scaffold resulted in
a steric repulsion between the gatekeeper residue and the
inhibitor’s central phenyl ring leading to a lower affinity against
the mutant form of cSrc.30 Derivatizing the quinazoline 6-amine
resulted in increased potency versus related nonmodified
structures (3f−i vs 3a−e). Except for 3c, equipped with a
primary amine, the whole set of 1,4-fused hybrid compounds
follows this general observation. Notably, there was no difference
in efficacy observed between 3f and 3h (both ∼1 nM),
illustrating that an additional interaction to Asp348 is not
obligatory for improved binding. Rather, the composition of
quinazoline-6-amide and meta-amine is responsible for the
observed effect.
Complex Crystal Structures of Type II Hybrids in cSrc.
To confirm the proposed general binding mode of our type II
hybrids and to study the relevance of the newly introduced
moieties we cocrystallized wild-type cSrc with 3g and 3h
(Figure 2). As anticipated by the modeling results, these hybrid
inhibitors were bound in a type II manner, accessing both
allosteric and ATP-binding pockets, thereby locking the kinase
in its inactive DFG-out conformation. Similar to the crystal
structure of the parent compound (2a), the quinazoline N1
generates a direct hydrogen bond to the peptide backbone of
Met341, located within the cSrc hinge region. The 1,4-fused
phenyl ring of the inhibitor is sandwiched between the gatekeeper
and Phe405 of the DFG motif, whereas the urea part is
F
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embedded within the groove formed by helix C (stabilized by two
hydrogen bonds to Glu310) and DFG motif (one hydrogen bond
to Asp404). An additional interaction was created by the pyrazolo
portion, which binds to the allosteric subpocket by forming a
water-mediated hydrogen bond to Asp404 (Figure 2). The newly
introduced 6-quinazoline substituents (4-(dimethylamino)-
butanoic acid and 3-(4-methylpiperazin-1-yl)propanoic acid)
contributed to the affinity by an additional hydrogen bond to
Asp348. A novel protein ligand interaction was created by the
inhibitor’s primary amino moiety via a polar contact to a small
polar subpocket, which is located outward of the allosteric site and
is comprised by a water molecule (W69) and polar amino acid
side chains (Lys295, Glu310, and the backbone of the DFG-motif,
Figure 2C).
Hybrid Compounds Inhibit Drug-Resistant Mutants
KIT V559D/T760I and Abl T315I. The results from activity-
based studies on KIT and Abl were highly similar to those
conducted with cSrc. Consistent with our previous studies,
kinase selectivity profiling showed clear evidence for KIT as a
possible target for our hybrid inhibitors.30 Within the kinase
domain of KIT, the same principal features are potentially
addressable by our newly designed binders because comparison
to cSrc and biochemical investigations revealed strong inhibi-
tion against its constitutively active (V559D) and concomitant
gatekeeper mutated (T670I) form. We observed IC50 values in
the low nanomolar range against KIT wild-type for 3b−d and
3f−i (2.6−7.6 nM) whereas the 1,3-fused inhibitors 4a and
4b were slightly less potent, eliciting values around 30 nM
(Table 2). Consistent with the previous findings on cSrc, the
more rigid compounds (4a,b) completely lost their inhibitory
activity against the V559D/T670I mutant form. However, the
activity of this double mutant was impeded by the bulk of the
1,4-fused compounds without significant loss of efficiency for
most representatives (3c and 3g−i, 2.7−9.2 nM) due to their
adaptive phenyl ring, which most likely rotates by 90° and
avoids a steric clash with the bulky gatekeeper (isoleucine).
Strikingly, we observed a strong analog effect for 6a and 6b
against KIT (1.0 and 4.5 nM), but the transfer of this effect
to the gatekeeper mutant form was not achieved for 6b,
which exhibited a 1000-fold loss of potency, while 6a revealed
essentially no effect.
The 1,3-fused hybrid compounds 4a,b showed a very high
affinity for the Abl wild-type kinase (IC50 = 9−23 nM) whereas
they completely failed to inhibit the T315I gatekeeper mutant
form (Table 2). In contrast, the 1,4-fused hybrid compounds
3a−d and 3f−h exhibited a significant inhibition of wild-type
Abl (IC50 = 42−157 nM) and predominantly retained their
potency toward Abl T315I (IC50 = 81−176 nM). As seen in
cSrc, fragments 5a,b and scaffolds without the pyrazolo moiety
6a,b showed no evidence of binding, suggesting that further
interactions of this unique moiety were required for binding to
Abl. Thus, our approach of using inhibitors with rotatable motifs
to overcome gatekeeper mutations in kinases was successful for
the in vitro inhibition of cSrc, KIT, and Abl (for biochemical
selectivity profile of 3d see Figure S3, Supporting Information).
Type II Inhibitors are Active on GIST Cell Lines
Expressing KIT T670I. We investigated inhibitory effects of
selected derivatives on human, untreated, metastatic GIST cells
harboring a 57 bp deletion of exon 11, which leads to expression
of constitutively activated KIT (GIST-T1).35 Additionally, we
examined the corresponding T670I gatekeeper mutated variant
(GIST-T1 T670I) (Figure 3A,B,D). We treated each type of
the cells for 72 h with 3b, 3c, 3d, 3f, 3h, and 6b (0−30 μM);
imatinib and ponatinib served as control compounds. All tested
compounds markedly reduced the number of viable GIST-T1
cells. In particular, 3b, 3d, and 6b, without the quinazoline
6-position substitution, accounted for the most significant effects
(GI50 = 11−17 nM). Substituted representatives (3f,h) as well as
3c, carrying a primary amine at the 6-position, were somewhat
less effective (GI50 = 105−402 nM). Treatment of imatinib-
resistant GIST-T1 T670I cells generated a similar effect, showing
the 1,4-fused quinazoline-pyrazolo urea hybrids to preserve their
antiproliferative effects. The observed efficacies were comparable
to those observed for GIST-T1 with 3b and 3d, which were
the most potent derivatives (GI50 = 24−28 nM), followed by 3c
(110 nM).
Notably, a strong inhibitory effect was also observed for 6b
with respect to GIST-T1 T670I (GI50 = 121 nM). We observed
an almost 8-fold discrepancy in the inhibition of the isolated
protein KIT V559D/T670I (IC50 = 923 nM) versus the
inhibition in the corresponding cell line by 6b. Compound 6b
may represent a slow binder, possessing a slow on- and off-rate
and therefore its true activity within a biochemical system may
not be fully evident. The difference in efficacy may be related to
the marginal incubation times with the isolated protein (0.5 h)
compared with cellular systems (72 h). A cellular system
represents an open equilibrium binding situation and a slow off-
rate could potentially lead to temporal target selectivity.36
Treatment of KIT-negative GIST-48B cells did not result in
decreased proliferation, indicating no incidence of generic
toxicity for the whole set of tested compounds with GI50 values
≥30 μM except 3c, 3f, and 3h, which did not elicit any anti-
proliferative effect at concentrations up to 30 μM (Figure 3C).
Therefore, the tested molecules without exception revealed an
improved off-target profile in GISTs compared with imatinib
(GI50 = 20 μM) and ponatinib (2.1 μM), a phase III clinical
candidate for treatment of CML.24
In order to study the immediate impact on cellular KIT
autophosphorylation and disruption of downstream signaling,
we monitored phosphorylation states of KIT Y703 (autophos-
phorylation site) and the related downstream targets Akt
(pS473) and MAPK (pT202/Y204) by Western blot analysis
(Figure 3E,F). Treatment with 3d and 3h reduced cellular
amounts of pKIT and simultaneously pAkt and pMAPK in
GIST-T1 in a concentration-dependent manner. Applying
70 nM 3d or 2000 nM 3h led to 50% abrogation of the total
amount of protein, confirming the direct effect of the hybrid
compounds on KIT. The different substitution pattern of 3d
and 3h led to different cellular performances (Figure 3A−D).
Compound 3d has no substituent on the quinazoline 6-
position, whereas 3h has an ethylenamide-linked methylpiper-
azine which apparently led to reduced efficacy by most likely
increasing both polarity and molecular weight. This observation
was accompanied by a simliar reduction of pKIT and downstream
signaling levels through pAkt and pMAPK caused by 3d and 3h at
distinct concentrations (50% reduction of pKIT at 35 nM and
400 nM, respectively) in the corresponding GIST-T1 T760I cells
expressing gatekeeper mutated KIT. However, these results
confirmed our initial hypothesis on clinically relevant gatekeeper
mutations to be overcome in a cellular system by equipping small
molecules with elements which incorporate rotational flexibility.
Additional experiments were performed in order to
investigate the inhibitory effect on Bcr-Abl and the respective
T315I gatekeeper mutated species in Ba/F3 cells (Figure S4,
Supporting Information). According to the biochemical
investigations on isolated kinase domains, the 1,4-fused inhibitors
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Figure 3. Dose−response curves of hybrid compounds on GIST cells expressing (A) constitutively active KIT (GIST-T1) as well as (B) the
gatekeeper mutated KIT T670I (GIST-T1 T670I) and (C) the KIT negative cell line GIST-48B after 72 h incubation with the respective
compounds; (D) GI50 values on GIST-T1, GIST-T1 T670I, and GIST-48B; (E,F) Western blot analyses after 24 h of incubation with doses of 3d,
3h, and imatinib (IM) in GIST-T1 and GIST-T1 T670I.
were less potent. Nevertheless 3b and 3d were following the same
trend reducing the viability of Ba/F3 Bcr-Abl cells (GI50 = 4.1 μM
and 4.3 μM) and furthermore retaining the antiproliferative effect
on the gatekeeper resistant mutation in Ba/F3 Bcr-Abl T315I
(GI50 = 3.5 μM and 1.6 μM). Comparatively to the GIST studies,
these results reflect the biochemical characterizations, which
indicated that the inhibitory effect on KIT is more significant as
compared with Abl.
additional studies using liposarcoma (LPS141), leiomyosarco-
ma (LMS676), and malignant peripheral nerve sheath tumor
cells (MPNST) were conducted (Figure S5, Supporting
Information). There was no significant effect observed for the
whole set of compounds. The most representative inhibitor,
3d, showed GI50’s of around 15.1 μM (LPS141), 25.7 μM
(LMS676), and 11.7 μM (MPNST), while its 6-position
extended analogue 3h did not significantly decrease the number
of viable cells up to inhibitor concentrations of 30 μM.
Interestingly, the marketed drug ponatinib reduced the viability
Cytotoxicity and Inhibition of Metabolic Enzymes. In
order to explore the hybrid compounds’ general cytotoxicity
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of the respective cell lines to 50% at concentrations ranging
from 0.1 to 0.8 μM. Next, we investigated the inhibitory impact
of 3d and 3h on five CYPs (1A2, 2C9, 2C19, 2D6, and 3A4), as
representative drug metabolizing enzymes and predictive
markers for potential adverse effects (Table S2, Supporting
Information). None of the tested cytochromes except 3A4
(IC50 = 14.8 μM and 1.95 μM for 3d and 3h) was remarkably
inhibited.
6-position (3b/d, 6b) was superior to the effect of compounds
bearing an extension (3f/h), suggesting that additional
interactions with Asn680 (located at the lip of the hinge region)
are not necessary at the cellular level, which agrees with the
concept of enthalpy−entropy compensation. Furthermore, the
introduction of polar moieties may affect the capability of the
compounds to pass through the cell membrane, which may result
in the generation of less significant cellular concentrations
and thereby reduced potency of the quinazoline 6-position
substituted compounds.
The presented hybrid derivatives exhibit enormous potential
in the therapeutic treatment of solid GISTs through addressing
gain of function mutants of KIT, and due to their central and
rotatable element they are similarly able to target the
gatekeeper mutated T670I species. A recently released study
by O’Hare et al. demonstrated that a linear triple bond as a
connecting structural element between type I and type III like
binders is likewise useful to overcome gatekeeper mutations in
Abl due to its decreased spatial arrangement.38 Derivatives
harboring this structural motif were shown to conserve their
inhibitory effect even against the T315I gatekeeper mutant
form in xenograft models, and one representative (ponatinib) is
now in phase III clinical trials as a pan-Bcr-Abl inhibitor for
CML treatment.24,38 Recently the FDA granted accelerated
approval for ponatinib in the treatment of CML with resistance
or intolerance to prior tyrosine kinase inhibitor therapy.
However, severe side effects with hepatotoxicity leading to
liver failure and death in some cases and 8−11% of treated
patients revealing arterial thrombosis, were observed in clinical
trials, indicating a rather complex pharmacology.39
In conclusion, our previously described quinazoline−pyrazo-
lourea hybrids were optimized by using methods of medicinal
chemistry. Using initial modeling studies and evaluating the
improved effect by biochemical characterizations, we successfully
obtained substantial transferability of the preliminarily deter-
mined binding mode to the clinically relevant kinases KIT and
Abl. Furthermore, their constitutively active variants and
gatekeeper mutants (T670I of KIT and T315I of Abl) were
significantly inhibited as shown in biochemical characterizations
using isolated kinase domains. Finally, compounds 3b, 3c, 3d, 3f,
3h, and 6b exhibit strong antiproliferative effects in cells derived
from solid tumors harboring constitutively active KIT (GIST-
T1) as well as gatekeeper mutated KIT T670I (GIST-T1 T670I)
variants and thus represent valuable starting points for future
medicinal chemistry endeavors.
DISCUSSION AND CONCLUSIONS
■
The emergence of drug resistance in kinases due to acquired
secondary mutations, with the most considerable mutations
affecting the gatekeeper residue, is a central challenge for
developing novel inhibitors for the long-term treatment of
cancer. Here, we present the development and optimization of
1,4-fused quinazoline−pyrazolourea hybrids by structure-based
design to compose and synthesize a focused library of
inhibitors. SAR studies showed an improved activity of hybrid
compounds 3f−h toward cSrc (IC50 ≈ 1 nM) compared with
initially published inhibitors. An enhanced effect toward cSrc
resulted from the newly introduced meta-oriented amino
moiety at the pyrazolo-phenyl ring whereas the formation of
an extra hydrogen bond to the kinase apparently was not crucial
for increased potency (Table 2, 3f vs 3h). Rather, the presence
of a meta-amine and an obligatory amide at the quinazoline
6-position led to improved binding effects. These findings can
be closely correlated to the enthalpy−entropy compensation
caused by the entropically disfavored fixation of the ligand, for
example in 3h, which compensates the favorable enthalpic
increase generated by the new hydrogen bond to Asp348
(Figure 2). The binding between the protein and the polar
moiety of the inhibitor contributes to the desolvation energy by
stripping away the hydration shell. In the case of compounds
3g−i, the surrounding tertiary amines represent an energetic
barrier that must be overcome.37 In addition, we achieved the
transfer of the inhibitory activity onto stem cell factor receptor
KIT (wild-type and V559D/T670I mutant, IC50 = 3−8 nM and
3−38 nM, respectively, for 3b−d, 3f−i), as well as Abl kinase
(wild-type and T315I mutant, IC50 = 40−157 nM and 81−561 nM,
respectively, for 3a−d,h). The preserved inhibitory effects with
respect to gatekeeper mutated kinases bearing bulky sub-
stituents on that position was likewise due to the spatial
arrangement of the rotatable 1,4-fused phenyl ring connecting
the inhibitor’s type I and type III parts. A 90° axial rotation of
this element, as shown for addressing the T338M gatekeeper
mutated form of cSrc,30 likewise accounted for the potent
inhibition of the corresponding mutated kinases KIT T670I
and Abl T315I.
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 anhydrous
In cellular characterizations, we further extended the hybrids’
impact from significant in vitro inhibition of wild-type KIT
as well as the gatekeeper and its simultaneously activating
mutant form (V559D/T670I) to addressing the relevant stem
cell factor receptor kinase species in human, untreated,
metastatic GIST-T1 cells and, particularly, the KIT T670I
positive cell line GIST-T1 T670I. We observed a significant
impact in reduction of viable GIST-T1 (GI50 = 11−17 nM for
3b/d, 6b) cells and moreover the inhibitory effect was
maintained in GIST-T1 T670I (GI50 = 24−121 nM for 3b/d, 6b)
cells. The latter observation underlines our successful approach
to generate hybrid compounds containing motifs with rota-
tional flexibility that can override the mutations of gatekeeper
residues in a cellular system. Notably, the inhibitory effect
for compounds lacking further substitutions at the quinazoline
reagents from commercial suppliers. H and 13C NMR spectra were
1
recorded on a Bruker Avance DRX 400 spectrometer at 400 and 101 MHz,
1
respectively. H chemical shifts are reported 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) or DMSO-d6 (2.50). 13C spectra are referenced to the
residual solvent signal: CDCl3 (77.16) or DMSO-d6 (39.52). 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
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(Pro Star 215) with a VP 250/21 nucleosil C18 PPN column from
Macherey-Nagel and monitored by UV at λ = 254 nm. All final compounds
were purified to ≥95% purity confirmed by LCMS analysis on LCQ
Advantage MAX (1200 series, Agilent) with Eclipse XDB-C18-column
(5 μM, 150 × 1.6 mm, Phenomenex).
3-tert-Butyl-1-(3-nitrophenyl)-1H-pyrazol-5-amine Hydrochloride
(7). A solution of m-nitrophenylhydrazine hydrochloride (2.00 g, 10.55
mmol) and pivaloylacetonitrile (1.53 g, 11.88 mmol) in dry EtOH
(22 mL) was treated with concentrated HCl (3 mL) and refluxed for
12 h at 90 °C. The precipitate was filtered off and dried under reduced
pressure before the crude was recrystallized from plain hexane yielding
2.94 g (9.91 mmol, 94%) of white crystals: 1H NMR (400 MHz,
CDCl3) δ 8.45 (t, J = 2.1 Hz, 1H), 8.09 (m, 2H), 7.73 (t, J = 8.2 Hz,
1H), 5.48 (bs, 2H), 5.47 (s, 1H), 1.23 (s, 9H); 13C NMR (101 MHz,
CDCl3) δ 162.12, 148.09, 147.96, 140.59, 130.48, 127.70, 119.59,
115.99, 88.22, 31.93, 30.03. HRMS (ESI-MS) calcd: 261.13460 for
C13H17N4O2 [M + H+]. Found: 261.13456.
into saturated bicarbonate solution followed by extraction with EtOAc
(3 × 20 mL). The combined organic layers were dried over Na2SO4, and
the volatiles were removed under reduced pressure. The crude was
purified by silica gel column chromatography.
tert-Butyl 4-(3-(3-tert-Butyl-1-(3-nitrophenyl)-1H-pyrazol-5-yl)-
ureido)phenylcarbamate (9). Compound 9 was prepared as des-
cribed in the general procedure using tert-butyl 4-aminophenylcarbamate
(1.43 g, 6.88 mmol), DMSO (25 mL), DIPEA (2.36 mL, 13.8 mmol),
and 8 (3 g, 6.88 mmol). The crude material was purified on silica gel
(20−50% EtOAc/petrol ether) to yield 2.07 g (4.19 mmol, 61%) of a
light yellow solid: 1H NMR (400 MHz, DMSO-d6) δ 8.23 (s, 1H), 7.98
(d, J = 8.0 Hz, 1H), 7.80 (s, 2H), 7.72 (d, J = 7.7 Hz, 1H), 7.44−7.40
(m, 1H), 7.10 (d, J = 8.4 Hz, 2H), 6.99 (d, J = 8.6 Hz, 2H), 6.58 (s,
1H), 6.38 (s, 1H), 1.45 (s, 9H), 1.28 (s, 9H); 13C NMR (101 MHz,
DMSO-d6) δ 163.56, 151.93, 148.79, 139.40, 134.82, 130.51, 130.02,
122.36, 119.33, 99.55, 94.83, 76.35, 75.17, 60.73, 32.74, 30.29, 26.26,
21.24, 14.32. HRMS (ESI-MS) calcd: 495.23504 for C25H31N6O3
[M + H+]. Found: 495.23445.
tert-Butyl 3-(3-(3-tert-Butyl-1-(3-nitrophenyl)-1H-pyrazol-5-yl)-
ureido)phenylcarbamate (10). Compound 10 was prepared as
described in the general procedure using tert-butyl 3-aminophenylcarba-
mate (300 mg, 0.69 mmol), DMSO (3 mL), DIPEA (236 μL, 1.38 mmol),
and 8 (144 mg, 0.69 mmol). The crude material was purified on silica gel
(0−0.5% MeOH/DCM) to yield 276 mg (0.56 mmol, 81%) of a light
yellow solid: 1H NMR (400 MHz, DMSO-d6) δ 9.32 (s, 1H), 9.05 (s, 1H),
8.46 (s, 1H), 8.36 (t, J = 2.1 Hz, 1H), 8.22 (dd, J = 8.2, 1.5 Hz, 1H), 8.08−
8.02 (m, 1H), 7.81 (t, J = 8.2 Hz, 1H), 7.57 (s, 1H), 7.11 (dd, J = 7.7, 4.5
Hz, 2H), 7.00−6.95 (m, 1H), 6.42 (s, 1H), 1.46 (s, 9H), 1.30 (s, 9H); 13C
NMR (101 MHz, DMSO-d6) δ 161.94, 152.70, 151.74, 148.14, 139.97,
139.61, 139.59, 137.80, 130.83, 129.57, 128.92, 121.42, 117.97, 112.28,
112.03, 108.07, 97.59, 79.01, 32.19, 30.07, 28.14. HRMS (ESI-MS) calcd:
495.23504 for C25H31N6O3 [M + H+]. Found: 495.23443.
2,2,2-Trichloroethyl 1-(3-nitrophenyl)-1H-pyrazol-5-ylcarbamate
(8). NaOH (1.13 g, 28.3 mmol) was added to a suspension of 7
(2.94 g, 11.3 mmol) in EtOAc (20 mL) and H2O (6 mL) at 0 °C.
The resulting reaction mixture was stirred for 30 min before 2,2,2-
trichloroethyl chloroformate (2.28 mL, 17.0 mmol) was added
dropwise. After an additional 30 min, the mixture was warmed to
room temperature and stirred for 3 h. The organic layer was separated
from the aqueous layer, which then was extracted with EtOAc (3 × 25 mL).
The combined organic layers were once washed with brine, dried over
Na2SO4, and concentrated under reduced pressure. Purification of
the crude by silica gel column chromatography (20% EtOAc/petrol
ether) lead to 4.79 g (11.0 mmol, 97%) of the desired product as a
1
brown resin: H NMR (400 MHz, CDCl3) δ 8.37 (s, 1H), 8.17 (dd,
J = 8.1, 1.2 Hz, 1H), 7.88 (dd, J = 8.1 Hz, 1H), 7.60−7.65 (m, 1H), 6.35
(s, 1H), 4.76 (s, 2H), 3.08 (s, 1H), 1.33 (s, 9H); 13C NMR (101 MHz,
CDCl3) δ 163.56, 148.76, 139.44, 134.81, 130.48, 130.00, 122.31, 119.29,
99.55, 94.84, 76.32, 75.14, 32.72, 30.28. HRMS (ESI-MS) calcd:
435.03881 for C16H18Cl3N4O4 [M + H+]. Found: 435.03874.
tert-Butyl 4-(3-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-
phenyl)-3-tert-butyl-1H-pyrazol-5-yl)ureido)phenylcarbamate (11,
2 steps). In a dry and argon-flushed flask, a solution of 9 (960 mg,
1.35 mmol) in EtOH (6 mL) was treated with ammoniumformate
(510 mg, 8.1 mmol) and 5% Pd on charcoal (297 mg, 0.14 mmol) for
1 h at 70 °C. After filtration of the reaction mixture over Celite, the
solvents were removed in vacuo. Recrystallization of the crude product
from hexane resulted in 608 mg (1.31 mmol, 97%) of a pink solid,
1-(3-tert-Butyl-1-(3-nitrophenyl)-1H-pyrazol-5-yl)-3-methylurea
(4a). A suspension of 8 (100 mg, 0.23 mmol), methylamine hydro-
chloride (155 mg, 230 mmol), and DIPEA (393 μL) in DMSO (2 mL)
was stirred for 5 h at room temperature. Saturated sodium-bicarbonate
solution was added, and the resulting mixture was extracted with EtOAc
(5 × 15 mL). The combined organic layers were dried over Na2SO4,
and the volatiles were removed in vacuo. The crude was purified on
silica gel using 0.5% MeOH/DCM which lead to 59 mg (0.19 mmol,
83%) of the desired product as a light yellow solid: 1H NMR
(400 MHz, DMSO-d6) δ 8.39 (s, 1H), 8.32 (t, J = 2.1 Hz, 1H), 8.19
(ddd, J = 8.2, 2.2, 0.8 Hz, 1H), 8.00 (ddd, J = 8.0, 1.9, 0.8 Hz, 1H),
7.78 (t, J = 8.2 Hz, 1H), 6.40−6.33 (m, 1H), 6.29 (s, 1H), 2.58 (d, J =
4.6 Hz, 3H), 1.28 (s, 9H); 13C NMR (101 MHz, DMSO-d6) δ 161.75,
155.33, 148.03, 139.84, 138.45, 130.65, 129.21, 121.06, 117.46, 98.23,
32.13, 30.03, 26.41. HRMS (ESI-MS) calcd: 318.15607 for
C15H20N5O3 [M + H+]. Found: 318.15611.
1-(1-(3-Aminophenyl)-3-tert-butyl-1H-pyrazol-5-yl)-3-methylurea
(4b). A solution of 4a (104 mg, 0.33 mmol) and ammonium formate
(124 mg, 1.97 mmol) in EtOH (7 mL) was treated with 5% Pd on charcoal
(70 mg, 33 μmol). The resulting reaction mixture was refluxed for 1 h at
90 °C before the catalyst was filtered off and the volatiles were removed
under reduced pressure. The crude was purified on silica gel (2%
MeOH/DCM) yielding 67 mg (0.23 mmol, 70%) of a white solid:
1H NMR (400 MHz, DMSO-d6) δ 8.09 (s, 1H), 7.10 (t, J = 7.9 Hz, 1H),
6.66 (t, J = 1.9 Hz, 1H), 6.60−6.52 (m, 2H), 6.43 (q, J = 4.1 Hz, 1H),
6.22 (s, 1H), 5.36 (s, 2H), 2.61 (d, J = 4.6 Hz, 3H), 1.25 (s, 9H); 13C
NMR (101 MHz, DMSO-d6) δ 159.99, 154.75, 149.62, 139.31, 137.83,
129.32, 112.63, 111.21, 109.77, 94.01, 31.94, 30.27, 26.21. HRMS (ESI-
MS) calcd: 288.18189 for C15H22N5O [M + H+]. Found: 288.18188.
General Procedure for the Preparation of N-Boc-Aminophenyl-3-
(3-tert-butyl-1-(3-nitrophenyl)-1H-pyrazol-5-yl)ureas (9, 10). DIPEA
was added to a solution of N-Boc-phenylenediamine in abs. DMSO and
stirred under argon atmosphere at room temperature for 5 min. After
addition of 8 in one portion, the resulting reaction mixture was stirred for
24 h at 60 °C. After cooling to room temperature, the mixture was poured
1
which was used without further purification: H NMR (400 MHz,
DMSO-d6) δ 9.29 (s, 1H), 9.19 (s, 1H), 8.62 (s, 1H), 7.30 (q, J = 9.1
Hz, 4H), 7.13−7.09 (m, 1H), 6.69−6.68 (m, 1H), 6.62−6.53 (m, 2H),
6.30 (s, 1H), 5.37 (s, 2H), 1.46 (s, 9H), 1.26 (s, 9H); 13C NMR (101
MHz, DMSO-d6) δ 160.94, 158.51, 153.68, 150.52, 148.16, 145.64,
140.02, 138.14, 134.99, 133.42, 130.27, 119.30, 113.64, 112.17, 110.73,
36.26, 32.83, 31.12, 29.02. HRMS (ESI-MS) calcd: 465.26087 for
C25H33N6O3 [M + H+]. Found: 465.26040.
NaHCO3 (336 mg, 4.0 mmol) and Fmoc-Cl (297 mg, 0.14 mmol)
were added to a suspension of the previously synthesized amine in
water/dioxane (12 mL/17 mL), and the resulting reaction mixture was
stirred overnight at room temperature. After addition of another 10 mL
of water, the mixture was extracted with EtOAc (5 × 30 mL). The
combined organic layers were dried over Na2SO4, and the volatiles
were removed under reduced pressure. The crude product was purified
on silica gel (2% MeOH/DCM) resulting in 833 mg (1.21 mmol,
76%) of a pink resin: 1H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H),
9.21 (s, 1H), 8.86 (s, 1H), 8.34 (s, 1H), 7.91 (d, J = 7.5 Hz, 2H), 7.75
(d, J = 7.5 Hz, 2H), 7.63 (s, 1H), 7.54 (bs, 1H), 7.44−7.40 (t, J =
7.3 Hz, 3H), 7.38−7.31 (m, 4H), 7.28 (d, J = 9.0 Hz, 2H), 7.13 (d, J =
7.5 Hz, 1H), 6.36 (s, 1H), 4.51 (d, J = 6.5 Hz, 2H), 4.31 (t, J = 6.5 Hz,
1H), 1.46 (s, 9H), 1.27 (s, 9H); 13C NMR (101 MHz, DMSO-d6) δ
160.78, 153.38, 152.84, 151.33, 143.72, 140.82, 140.01, 138.80, 137.46,
134.80, 134.12, 133.80, 129.60, 127.71, 127.15, 125.13, 120.22, 118.64,
118.22, 114.38, 94.37, 91.87, 78.79, 65.68, 46.60, 32.04, 30.21, 28.17.
HRMS (ESI-MS) calcd: 687.32894 for C40H43N6O5 [M + H+]. Found:
687.32825.
(9H-Fluoren-9-yl)methyl 3-(3-tert-Butyl-5-(3-(4-(6-nitroquinazolin-4-
ylamino)phenyl)ureido)-1H-pyrazol-1-yl)phenylcarbamate (12, 2 steps).
A solution of 11 (814 mg, 1.19 mmol) in dioxane (2 mL) was treated
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with HCl (8 mL, 4 M in dioxane) for 2 h at rt. After the reaction was
completed, the volatiles were removed under reduced pressure and
subsequently EtOAc (10 mL) and saturated sodium bicarbonate
solution (10 mL) were added. The aqueous layer was extracted with
EtOAc (3 × 20 mL), and the combined organic layers were dried
over Na2SO4. The solvent was removed in vacuo leading to 686 mg
(1.17 mmol, 98%) of a light yellow solid, which was used without
for 10 min at 0 °C and for 1 h at room temperature. The volatiles were
removed in vacuo, and the remaining solid was dissolved in EtOAc and
saturated sodium bicarbonate solution. The aqueous layer was extracted
with EtOAc (3 × 15 mL), and the combined organic layers were dried
over Na2SO4. After that, the volatiles were evaporated under reduced
pressure leading to 73 mg of a yellow-green crude product, which was
used without further purification.
1
further purification: H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H),
The remaining solid (69 mg) was treated with morpholine (1 mL,
30% in DMF) for 15 min at room temperature. Afterward, the solvents
were removed under reduced pressure, and the crude was purified
on silica gel using 10% MeOH/DCM. The resulting yellow solid
was dissolved in THF and filtered through a membrane filter. After
removal of the solvent under reduced pressure, 32 mg (56.8 μmol,
8.50 (s, 1H), 8.22 (s, 1H), 7.91 (d, J = 7.5 Hz, 2H), 7.76 (d, J = 7.4
Hz, 2H), 7.63 (s, 1H), 7.55 (s, 1H), 7.47−7.31 (m, 6H), 7.12 (d, J =
7.6 Hz, 1H), 7.03 (d, J = 8.7 Hz, 2H), 6.49 (d, J = 8.7 Hz, 2H), 6.33
(s, 1H), 4.84 (s, 2H), 4.51 (d, J = 6.5 Hz, 2H), 1.26 (s, 9H); 13C NMR
(101 MHz, DMSO-d6) δ 160.73, 153.38, 151.55, 144.12, 143.73,
140.82, 139.99, 138.87, 137.81, 129.57, 128.27, 127.72, 127.15, 125.14,
120.46, 120.22, 118.20, 114.36, 114.14, 94.09, 91.54, 65.69, 46.60,
32.03, 30.22. HRMS (ESI-MS) calcd: 587.27652 for C35H35N6O3
[M + H+]. Found: 587.27588.
1
83%) of the desired yellow solid was isolated: H NMR (400 MHz,
DMSO-d6) δ 10.20 (s, 1H), 9.73 (s, 1H), 9.14 (s, 1H), 8.69 (s, 1H),
8.45 (s, 1H), 8.37 (s, 1H), 7.81 (dd, J = 9.0, 1.9 Hz, 1H), 7.72 (d, J =
8.9 Hz, 1H), 7.68 (d, J = 8.9 Hz, 2H), 7.43 (d, J = 8.9 Hz, 2H), 7.18−
7.14 (m, 1H), 6.70 (d, J = 1.9 Hz, 1H), 6.61 (dd, J = 8.0, 1.7 Hz, 2H),
6.37 (s, 1H), 5.42 (s, 2H), 2.41 (q, J = 7.5 Hz, 2H), 1.27 (s, 9H), 1.14
(t, J = 7.5 Hz, 3H); 13C NMR (101 MHz, DMSO-d6) δ 172.12,
160.23, 157.91, 157.50, 153.29, 151.27, 149.81, 146.35, 139.08, 137.22,
136.76, 135.32, 133.73, 129.58, 128.21, 123.30, 118.12, 115.40, 112.97,
111.92, 111.50, 110.02, 93.54, 32.02, 30.30, 29.39, 9.66. HRMS (ESI-
MS) calcd: 564.28300 for C31H34N9O2 [M + H+]. Found: 564.28278.
General Procedure for the Preparation of 1,4-Fused Hybrid
Derivatives (3g−i). (COCl)2 and catalytic amounts of DMF were
added to a suspension of the appropriate acid of choice in THF or
DCM under an argon atmosphere. The resulting mixture was stirred for
2 h at 30−35 °C before 13, dissolved in 1−2 mL of NMP, was added in
one portion. After another hour of stirring at room temperature, the
reaction was completed. Subsequently saturated sodium bicarbonate
solution was added and the aqueous layer extracted with EtOAc (3 ×
20 mL). The combined organic layers were dried over Na2SO4 and the
volatiles removed under reduced pressure. Treatment of the crude with
a solution of 20−25% morpholine in THF or THF/DMF (4:1) for
30−60 min led to the removal of the Fmoc protecting group. After
evaporation of the solvents in vacuo, the crude was purified by HPLC.
N-(4-(4-(3-(1-(3-Aminophenyl)-3-tert-butyl-1H-pyrazol-5-yl)-
ureido)phenylamino)quinazolin-6-yl)-4-(dimethylamino)-
butanamide (3g). Compound 3g was prepared as described in the
general procedure using THF (3 mL), 4-(dimethylamino)butanoic
acid (100 mg, 597 μmol), (COCl)2 (52.3 μL, 597 μmol), DMF (two
drops), and 3e (50 mg, 68.5 μmol) in 1 mL of NMP. Deprotection
was performed by treating the crude material with 20% morpholine in
THF (2.4 mL) for 40 min. The volatiles were removed under reduced
pressure, and the crude product was purified by preparative HPLC
(H2O/MeCN + 0.1% TFA). The combined, product containing
fractions were concentrated under reduced pressure and subsequently
basified with saturated sodium bicarbonate solution. After extraction of
the aqueous layer with EtOAc (3 × 15 mL), drying of the combined
organic layers over Na2SO4, and evaporation of the residual solvent in
vacuo, 27 mg (43.5 μmol, 64%) of the desired product was isolated as
A solution of the previously synthesized amino hydrochloride
(739 mg, 1.26 mmol) in DCM (33 mL) under argon atmosphere was
treated with DIPEA (1 mL, 5.84 mmol) for 10 min at rt. Thereafter
freshly prepared 4-chloro-6-nitroquinazolinol (317 mg, 1.51 mmol)
was added, and the resulting reaction mixture was stirred for 22 h at rt.
After addition of saturated sodium bicarbonate solution and separation
of the phases, the aqueous layer was extracted with DCM (3 × 50 mL).
The combined organic layers were dried over Na2SO4, and the
volatiles were removed under reduced pressure. The crude material
was purified on silica gel (1−4% MeOH/DCM) yielding 743 mg (0.97
mmol, 77%) of the desired product as a yellow-orange solid: 1H NMR
(400 MHz, DMSO-d6) δ 10.41 (s, 1H), 9.98 (s, 1H), 9.64 (s, 1H),
9.07 (s, 1H), 8.66 (s, 1H), 8.54 (dd, J = 9.2, 2.3 Hz, 1H), 8.44 (s, 1H),
7.95−7.84 (m, 3H), 7.74 (dd, J = 14.2, 8.2 Hz, 4H), 7.67 (s, 1H), 7.55
(s, 1H), 7.47 (d, J = 8.9 Hz, 2H), 7.43−7.40 (m, 3H), 7.34 (t, J = 7.1
Hz, 2H), 7.16 (d, J = 7.7 Hz, 1H), 6.40 (s, 1H), 4.51 (d, J = 6.5 Hz,
2H), 4.31 (t, J = 6.5 Hz, 1H), 1.28 (s, 9H); 13C NMR (101 MHz,
DMSO-d6) δ 160.80, 158.68, 157.85, 153.39, 153.13, 151.39, 144.42,
143.72, 140.81, 140.03, 138.82, 137.35, 136.13, 132.63, 129.62, 129.41,
127.71, 127.14, 126.56, 125.14, 123.65, 120.84, 120.21, 118.26, 118.23,
118.19, 114.39, 94.67, 65.70, 60.85, 46.60, 32.07, 30.22. HRMS (ESI-
MS) calcd: 760.29904 for C43H38N9O5 [M + H+]. Found: 760.30027.
(9H-Fluoren-9-yl)methyl 3-(5-(3-(4-(6-Aminoquinazolin-4-ylamino)-
phenyl)ureido)-3-tert-butyl-1H-pyrazol-1-yl)phenylcarbamate (3e).
Iron turnings (269 mg, 4.56 mmol) were activated, by treating them
two times with 1 N HCl for 20 min at room temperature, and added to
a suspension of 12 (434 mg, 0.57 mmol) in EtOH (40 mL) and AcOH
(0.13 mL, 2.28 mmol). The resulting reaction mixture was refluxed for
2.5 h. After removal of the iron turnings, the remaining solution was
basified with aqueous NH3. The solvents were removed under reduced
pressure, and the residue was dissolved with EtOAc and saturated
sodium bicarbonate solution. Subsequently, the aqueous layer was
extracted with EtOAc (10 × 40 mL), and the combined organic layers
were dried over Na2SO4. The crude was purified on silica gel (4−6%
MeOH/DCM) leading to 316 mg (0.43 mmol, 75%) of an off yellow
1
1
solid: H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 9.29 (s, 1H),
a light green solid: H NMR (600 MHz, DMSO-d6) δ 10.22 (s, 1H),
8.99 (s, 1H), 8.42 (s, 1H), 8.28 (s, 1H), 7.90 (d, J = 7.5 Hz, 2H),
7.80−7.70 (m, 4H), 7.66 (s, 1H), 7.55 (s, 1H), 7.51 (d, J = 8.8 Hz,
1H), 7.47−7.38 (m, 5H), 7.37−7.33 (m, 3H), 7.22 (dd, J = 8.9, 2.0 Hz,
1H), 7.16 (d, J = 7.8 Hz, 1H), 6.39 (s, 1H), 5.56 (s, 2H), 4.51 (d, J =
6.5 Hz, 2H), 4.32 (t, J = 6.5 Hz, 1H),1.28 (s, 9H); 13C NMR (101 MHz,
DMSO-d6) δ 160.80, 156.04, 153.40, 151.40, 149.97, 147.16, 143.73,
142.36, 140.82, 140.03, 138.83, 137.47, 134.68, 134.37, 133.68, 129.62,
128.55, 127.72, 127.15, 125.14, 123.43, 122.57, 120.22, 118.26, 116.90,
116.59, 114.41, 101.14, 94.49, 65.69, 46.61, 32.06, 30.23. HRMS (ESI-
MS) calcd: 730.32486 for C43H40N9O3 [M + H+]. Found: 730.32501.
N-(4-(4-(3-(1-(3-Aminophenyl)-3-tert-butyl-1H-pyrazol-5-yl)-
ureido)phenylamino)quinazolin-6-yl)propionamide (3f). A solution
of 3e (50 mg, 68.5 μmol) in abs. THF (4 mL) under argon atmosphere
was cooled to 0 °C before DIPEA (23.4 μL, 137 μmol) was added.
Subsequently a diluted solution of propionylchloride (7.45 μL,
82.2 μmol) in THF (7.45 mL) was added dropwise over 45 min.
After addition was completed, the resulting reaction mixture was stirred
9.70 (s, 1H), 9.12 (s, 1H), 8.67 (d, J = 1.6 Hz, 1H), 8.45 (s, 1H), 8.36
(s, 1H), 7.82 (d, J = 2.1 Hz, 1H), 7.72 (d, J = 8.9 Hz, 1H), 7.69 (d, J =
8.9 Hz, 2H), 7.45−7.40 (m, 2H), 7.16 (t, J = 7.9 Hz, 1H), 6.70 (t, J =
2.1 Hz, 1H), 6.62−6.61(t, J = 2.4 Hz, 1H), 6.61−6.60 (t, J = 2.2 Hz,
1H), 6.36 (s, 1H), 5.39 (s, 2H), 2.42 (t, J = 7.4 Hz, 2H), 2.37 (t, J =
7.4 Hz, 2H), 2.22 (s, 6H), 1.79 (p, J = 7.4 Hz, 2H), 1.27 (s, 9H); 13C
NMR (101 MHz, DMSO-d6) δ 171.14, 160.17, 157.46, 153.24, 151.25,
149.73, 146.34, 139.07, 137.17, 136.69, 135.29, 133.70, 129.49, 128.15,
124.87, 123.25, 118.10, 115.35, 112.92, 111.88, 111.45, 109.99, 93.55,
58.23, 44.79, 31.95, 30.40, 30.24, 22.59. HRMS (ESI-MS) calcd:
621.34085 for C34H41N10O2 [M + H+]. Found: 621.34078.
N-(4-(4-(3-(1-(3-Aminophenyl)-3-tert-butyl-1H-pyrazol-5-yl)-
ureido)phenylamino)quinazolin-6-yl)-3-(4-methylpiperazin-1-yl)-
propanamide·TFA (3h). Compound 3h was prepared as described in
the general procedure using DCM (3 mL), 3-(4-methylpiperazin-1-
yl)propanoic acid (35 mg, 200 μmol), (COCl)2 (17.5 μL, 200 μmol),
DMF (three drops), and 3e (50 mg, 68.5 μmol) in NMP (1.5 mL).
K
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Treatment of the crude with a solution of 20% morpholine in THF/
DMF (5 mL, 4:1) for 1 h led to the removal of the Fmoc protecting
group. After evaporation of the volatiles under reduced pressure, the
crude product was purified by preparative HPLC (H2O/MeCN + 0.1%
TFA) yielding 23 mg (29.6 μmol, 43%) of the desired product as a yellow
The thionyl chloride was evaporated under reduced pressure, and the
remaining residue was dried under high vacuum and used without
further purification.
1-(3-tert-Butyl-1-(3-nitrophenyl)-1H-pyrazol-5-yl)-3-(4-(6-nitro-
quinazolin-4-ylamino)phenyl)urea (3a). A solution of the amino
hydrochloride 13 (77 mg, 179 μmol) and DIPEA (153 μL, 894 μmol) in
DCM (10 mL) was stirred for 5 min at room temperature. Then freshly
prepared 4-chloro-6-nitroquinazoline (55 mg, 215 μmol) was added, and
the resulting reaction mixture was stirred for 23 h at room temperature.
After addition of saturated sodium bicarbonate solution the aqueous layer
was extracted with EtOAc (3 × 10 mL) and the combined organic layers
were dried over Na2SO4. The volatiles were removed in vacuo, and the
purification of the crude on silica gel (1−4% MeOH/DCM) lead to
1
solid: H NMR (400 MHz, DMSO-d6) δ 11.53 (s, 1H), 10.85 (s, 1H),
9.46 (s, 1H), 8.98 (s, 1H), 8.85 (s, 1H), 8.72 (s, 1H), 8.07 (d, J = 9.1 Hz,
1H), 7.89 (d, J = 9.0 Hz, 1H), 7.55 (q, J = 9.2 Hz, 4H), 7.35 (t, J = 8.0
Hz, 2H), 7.09 (s, 1H), 7.05 (d, J = 8.1 Hz, 1H), 6.92 (d, J = 7.9 Hz, 2H),
6.37 (s, 1H), 3.34 (s, 2H), 3.34 (bs, 8H), 2.94−2.87 (m, 2H), 2.86 (s,
3H), 1.28 (s, 9H); 13C NMR (101 MHz, DMSO-d6) δ 168.87, 160.75,
159.31, 159.16, 158.80, 158.45, 158.10, 151.72, 149.70, 139.38, 138.75,
138.19, 137.17, 134.60, 130.68, 129.97, 125.51, 120.58, 118.12, 117.45,
116.54, 114.54, 113.97, 112.74, 95.77, 51.49, 50.19, 48.58, 41.99, 32.09,
31.44, 30.22. HRMS (ESI-MS) calcd: 662.36740 for C36H44N11O2
[M + H+]. Found: 662.36721.
1
45.1 mg (79.0 μmol, 44%) of an orange solid: H NMR (400 MHz,
DMSO-d6): δ 10.41 (s, 1H), 9.63 (s, 1H), 9.11 (s, 1H), 8.65 (s, 1H), 8.60
(s, 1H), 8.54 (dd, J = 9.2, 2.4 Hz, 1H), 8.39 (s, 1H), 8.23 (d, J = 8.1 Hz,
1H), 8.07 (d, J = 8.1 Hz, 1H), 7.90 (d, J = 9.1 Hz, 1H), 7.82 (t, J = 8.1
Hz, 1H), 7.70 (d, J = 8.7 Hz, 2H), 7.45 (d, J = 8.8 Hz, 2H), 6.43 (s, 1H),
1.31 (s, 9H); 13C NMR (101 MHz, DMSO-d6) δ 161.99, 158.79, 157.89,
153.15, 152.16, 148.17, 144.48, 139.73, 137.79, 136.06, 132.81, 130.88,
129.58, 129.44, 126.61, 123.74, 121.46, 120.87, 118.59, 117.89, 98.20,
35.46, 32.25, 30.11. HRMS (ESI-MS) calcd: 568.20514 for C28H26N9O5
[M + H+]. Found: 568.20480.
N-(4-(4-(3-(1-(3-Aminophenyl)-3-tert-butyl-1H-pyrazol-5-yl)ureido)-
phenylamino)quinazolin-6-yl)-3-morpholinopropanamide·TFA (3i).
Compound 3i was prepared as described in the general procedure using
DCM (3 mL), 3-morpholinopropanoic acid (50 mg, 256 μmol), (COCl)2
(22.4 μL, 256 μmol), DMF (three drops), and 3e (50 mg, 68.5 μmol) in
2 mL of NMP. Treatment of the crude with a solution of 25% morpholine
in THF (2.5 mL) for 30 min led to Fmoc-deprotection. After evaporation
of the volatiles under reduced pressure, the crude product was purified by
preparative HPLC (H2O/MeCN + 0.1% TFA) yielding 37 mg (48.5 μmol,
General Procedure for the Preparation of 1,3- And 1,4-Fused
Nitro-Substituted Hybrid Compounds (3b, 4a). A suspension of 13
or 14 and freshly prepared 4-chloroquinazoline in 2-propanol was stirred
for 3 h at 75 °C refluxing temperature. An excess of saturated sodium
bicarbonate solution was added to the chilled reaction batch followed by
extraction of the aqueous layer with EtOAc (4 × 20 mL). The combined
organic layers were dried over Na2SO4, and the solvent was removed under
reduced pressure before the crude material was purified on silica gel.
1-(3-tert-Butyl-1-(3-nitrophenyl)-1H-pyrazol-5-yl)-3-(4-(quinazolin-
4-ylamino)phenyl)urea (3b). Compound 3b was prepared as described
in the general procedure using 13 (137 mg, 0.32 mmol), 4-
chloroquinazoline (116 mg, 0.70 mmol), and 2-propanol (6 mL). Silica
gel column chromatography was performed using 2−4% MeOH/DCM
and lead to 80 mg (0.15 mmol, 47%) of the desired product as white
solid: 1H NMR (400 MHz, DMSO-d6) δ 9.75 (s, 1H), 9.06 (s, 1H), 8.58
(s, 1H), 8.56−8.50 (m, 2H), 8.40 (t, J = 2.1 Hz, 1H), 8.23 (ddd, J = 8.3,
2.2, 0.7 Hz, 1H), 8.11−8.04 (m, 1H), 7.84 (dt, J = 14.9, 4.5 Hz, 2H), 7.77
(d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.9 Hz, 2H), 7.66−7.59 (m, 1H), 7.43
(d, J = 8.9 Hz, 2H), 6.44 (s, 1H), 1.31 (s, 9H); 13C NMR (101 MHz,
DMSO-d6) δ 161.89, 157.73, 154.59, 152.07, 149.57, 148.12, 139.68,
137.80, 135.32, 133.59, 132.90, 130.81, 129.53, 127.73, 126.12, 123.28,
122.91, 121.37, 118.56, 117.85, 115.10, 98.02, 32.18, 30.06. HRMS (ESI-
MS) calcd: 523.22006 for C28H27N8O3 [M + H+]. Found: 523.21936.
1-(3-tert-Butyl-1-(3-nitrophenyl)-1H-pyrazol-5-yl)-3-(3-(quinazolin-
4-ylamino)phenyl)urea (4a). Compound 4a was prepared as
described in the general procedure using 14 (135 mg, 0.31 mmol),
4-chloroquinazoline (130 mg, 0.79 mmol), and 2-propanol (6 mL).
Silica gel column chromatography was performed using 1−2%
MeOH/DCM yielding 106 mg (0.20 mmol, 65%) of the desired
1
71%) of the desired product as a yellow solid: H NMR (400 MHz,
DMSO-d6) δ 11.56 (s, 1H), 10.92 (s, 1H), 9.52 (s, 1H), 8.98 (s, 1H), 8.85
(s, 2H), 8.08 (dd, J = 9.1, 1.5 Hz, 1H), 7.90 (d, J = 9.0 Hz, 1H), 7.55 (q,
J = 9.3 Hz, 5H), 7.44 (t, J = 8.0 Hz, 2H), 7.27 (d, J = 4.7 Hz, 2H), 7.07
(d, J = 7.9 Hz, 1H), 6.38 (s, 1H), 4.01 (d, J = 11.2 Hz, 2H), 3.69 (t, J =
11.1 Hz, 2H), 3.49 (t, J = 7.2 Hz, 4H), 3.17 (d, J = 9.7 Hz, 2H), 2.99 (t, J =
7.2 Hz, 2H), 1.29 (s, 9H); 13C NMR (101 MHz, DMSO-d6) δ 168.35,
161.03, 159.37, 159.19, 158.83, 158.47, 158.11, 151.93, 149.72, 139.55,
138.72, 138.28, 137.30, 134.56, 130.69, 130.20, 129.33, 125.57, 120.55,
118.19, 118.09, 117.32, 115.28, 114.41, 113.98, 112.90, 96.55, 63.43, 51.85,
51.45, 32.14, 30.21. HRMS (ESI-MS) calcd: 649.33576 for C35H41N10O3
[M + H+]. Found: 649.33563.
General Procedure for the Preparation of Aminophenyl-3-(3-tert-
butyl-1-(3-nitrophenyl)-1H-pyrazol-5-yl)urea Hydrochlorides (13, 14).
The corresponding N-Boc-protected molecules 9 and 10 were treated
with 4 M HCl in dioxane for about 45 min at room temperature. The
volatiles were removed in vacuo, giving the desired product, which was
further used without further purification.
1-(4-Aminophenyl)-3-(3-tert-butyl-1-(3-nitrophenyl)-1H-pyrazol-
5-yl)urea Hydrochloride (13). Compound 13 was prepared as described
in the general procedure using 9 (100 mg, 200 μmol) and 4 M HCl in
1
dioxane (3 mL) to obtain 85 mg (197 μmol, 99%) of a fawn solid: H
NMR (400 MHz, DMSO-d6) δ 10.21 (s, 2H), 9.86 (s, 1H), 9.16 (s, 1H),
8.36 (s, 1H), 8.20 (d, J = 8.1 Hz, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.80−7.76
(m, 1H), 7.50 (d, J = 8.8 Hz, 2H), 7.29 (d, J = 8.7 Hz, 2H), 6.41 (s, 1H),
1.30 (s, 9H); 13C NMR (101 MHz, DMSO-d6) δ 161.93, 152.27, 148.12,
139.56, 139.38, 137.54, 130.81, 129.44, 125.21, 123.84, 121.38, 118.88,
117.86, 98.24, 32.20, 30.05. HRMS (ESI-MS) calcd: 395.18262 for
C20H23N6O3 [M + H+]. Found: 395.18221.
1
product as light yellow solid: H NMR (400 MHz, DMSO-d6) δ 9.79
(s, 1H), 9.15 (s, 1H), 8.59 (t, J = 8.7 Hz, 3H), 8.39 (t, J = 2.0 Hz, 1H),
8.23 (dd, J = 8.2, 1.5 Hz, 1H), 8.07 (dd, J = 8.1, 1.1 Hz, 1H), 7.99 (s,
1H), 7.89−7.76 (m, 3H), 7.67−7.60 (m, 1H), 7.49 (d, J = 7.9 Hz,
1H), 7.31−7.20 (m, 2H), 6.44 (s, 1H), 1.30 (s, 9H); 13C NMR (101
MHz, DMSO-d6) δ 161.92, 157.78, 154.42, 151.91, 149.62, 148.14,
139.64, 139.55, 139.41, 137.77, 133.04, 130.83, 129.57, 128.72, 127.77,
126.27, 123.08, 121.41, 117.95, 116.55, 115.19, 113.88, 112.44, 97.75,
32.19, 30.06. HRMS (ESI-MS) calcd: 523.22006 for C28H27N8O3
[M + H+]. Found: 523.21933.
1-(3-Aminophenyl)-3-(3-tert-butyl-1-(3-nitrophenyl)-1H-pyrazol-
5-yl)urea Hydrochloride (14). Compound 14 was prepared as
described in the general procedure using 10 (113 mg, 230 μmol)
and 4 M HCl in dioxane (5 mL) to obtain 98 mg (228 μmol, 99%) of
1
a fawn solid: H NMR (400 MHz, DMSO-d6) δ 10.27 (s, 2H), 10.00
(s, 1H), 9.19 (s, 1H), 8.36 (s, 1H), 8.20 (dd, J = 8.2, 1.3 Hz, 1H), 8.07
(dd, J = 8.1, 1.0 Hz, 1H), 7.78 (t, J = 8.2 Hz, 1H), 7.62 (s, 1H), 7.40−
7.31 (m, 2H), 6.97 (dd, J = 6.5, 2.1 Hz, 1H), 6.41 (s, 1H), 1.30 (s, 9H);
13C NMR (101 MHz, DMSO-d6) δ 161.97, 152.20, 148.15, 140.71,
139.58, 137.45, 132.05, 130.85, 130.17, 129.40, 121.41, 117.86, 117.43,
116.57, 112.52, 98.26, 32.22, 30.07. HRMS (ESI-MS) calcd: 395.18262
for C20H23N6O3 [M + H+]. Found: 395.18182.
4-Chloro-6-nitroquinazoline or 4-Chloroquinazoline. A two-neck
flask was flushed with argon and charged with 6-nitroquinazolin-4-ol
or quinazolin-4-ol and thionyl chloride. A catalytic amount of DMF
was added, and the reaction mixture was refluxed at 80 °C overnight.
1-(1-(3-Aminophenyl)-3-tert-butyl-1H-pyrazol-5-yl)-3-(4-(6-amino-
quinazolin-4-ylamino)phenyl)urea (3c). A solution of 3a (79 mg, 0.14
mmol) in EtOH (5 mL) was treated with ammonium formate (52 mg,
0.82 mmol) and 5% Pd on charcoal (29 mg, 14 μmol) for 2 h at reflux.
The catalyst/reaction mixture was filtered over Celite, and the filtrate
was concentrated in vacuo. The crude material was purified on
silica gel (4% MeOH/DCM) yielding 48.3 mg (0.1 mmol, 71%) of a
1
yellow-orange solid: H NMR (400 MHz, DMSO-d6) δ 9.29 (s, 1H),
L
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9.18 (s, 1H), 8.41 (s, 1H), 8.29 (s, 1H), 7.74 (d, J = 8.9 Hz, 2H), 7.51
(d, J = 8.8 Hz, 1H), 7.42 (d, J = 8.9 Hz, 2H), 7.36 (s, 1H), 7.23 (dd,
J = 8.8, 2.0 Hz, 1H), 7.18−7.14 (m, 1H), 6.72 (s, 1H), 6.62 (d, J =
8.1 Hz, 2H), 6.37 (s, 1H), 5.55 (s, 2H), 5.42 (s, 2H), 1.28 (s, 9H);
13C NMR (101 MHz, DMSO-d6) δ 160.24, 156.07, 151.36, 150.04,
149.79, 147.15, 142.46, 139.11, 137.25, 134.82, 134.32, 129.57, 128.61,
123.44, 122.64, 118.18, 116.61, 112.97, 111.50, 110.02, 101.19, 93.67,
32.03, 30.31. HRMS (ESI-MS) calcd: 508.25678 for C28H30N9O
[M + H+]. Found: 508.25641.
General Procedure for the Preparation of 1,3- And 1,4-Fused
Amino-Substituted Hybrid Compounds (3d, 4b). A suspension of 3b
or 4a and SnCl2 in EtOH was stirred for 1.5−3 h at 70 °C. The
reaction mixture was concentrated in vacuo to a tenth of the starting
volume. Subsequently water was added, and the resulting solution was
extracted with EtOAc (7 × 30 mL). The combined organic layers were
dried over Na2SO4, and the volatiles were removed under reduced
pressure before the crude material was purified on silica gel.
1-(1-(3-Aminophenyl)-3-tert-butyl-1H-pyrazol-5-yl)-3-(4-(quina-
zolin-4-ylamino)phenyl)urea (3d). Compound 3d was prepared as
described in the general procedure using 3b (84 mg, 0.16 mmol),
SnCl2 (61 mg, 0.32 mmol), and EtOH (7 mL). Silica gel column
chromatography was performed (1−4% MeOH/DCM) resulting in
47 mg (0.10 mmol, 63%) of the desired product as a yellow solid:
1H NMR (400 MHz, DMSO-d6) δ 9.77 (s, 1H), 9.14 (s, 1H), 8.57−
8.51 (m, 2H), 8.36 (s, 1H), 7.85 (t, J = 7.6 Hz, 1H), 7.77 (d, J = 8.2
Hz, 1H), 7.73 (d, J = 8.9 Hz, 2H), 7.65−7.59 (m, 1H), 7.45 (d, J = 8.9
Hz, 2H), 7.16 (t, J = 7.9 Hz, 1H), 6.69 (t, J = 1.9 Hz, 1H), 6.64−6.57
(m, 2H), 6.37 (s, 1H), 5.46 (s, 2H), 1.27 (s, 9H); 13C NMR (101
MHz, DMSO-d6) δ 160.20, 157.74, 154.57, 151.21, 149.79, 149.44,
139.05, 137.18, 135.57, 133.34, 132.95, 129.55, 127.63, 126.17, 123.37,
122.94, 118.12, 115.09, 112.95, 111.47, 110.01, 93.47, 32.00, 30.28.
HRMS (ESI-MS) calcd: 493.24588 for C28H29N8O [M + H+]. Found:
493.24541.
1-(1-(3-Aminophenyl)-3-tert-butyl-1H-pyrazol-5-yl)-3-(3-(quina-
zolin-4-ylamino)phenyl)urea (4b). Compound 4b was prepared as
described in the general procedure using 4a (145 mg, 0.28 mmol), SnCl2
(156 mg, 0.82 mmol), and EtOH (10 mL). Silica gel column
chromatography was performed (1−4% MeOH/DCM) resulting in 82
mg (0.17 mmol, 61%) of the desired product as a white solid: 1H NMR
(400 MHz, DMSO-d6) δ 9.80 (s, 1H), 9.21 (s, 1H), 8.61−8.57 (m, 2H),
8.39 (s, 1H), 8.03 (s, 1H), 7.87 (t, J = 7.6 Hz, 1H), 7.79 (d, J = 8.1 Hz,
1H), 7.64 (t, J = 7.6 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.29 (t, J = 8.0 Hz,
1H), 7.23 (d, J = 8.3 Hz, 1H), 7.16 (t, J = 7.9 Hz, 1H), 6.69 (t, J = 1.9 Hz,
1H), 6.63−6.58 (m, 2H), 6.37 (s, 1H), 5.45 (s, 2H), 1.26 (s, 9H); 13C
NMR (101 MHz, DMSO-d6) δ 160.21, 157.78, 154.42, 151.10, 149.80,
149.60, 139.59, 139.57, 139.02, 137.13, 133.05, 129.58, 128.75, 127.75,
126.27, 123.09, 116.29, 115.20, 113.52, 112.97, 112.05, 111.50, 110.02,
93.34, 31.99, 30.27. HRMS (ESI-MS) calcd: 493.24588 for C28H29N8O
[M + H+]. Found: 493.24543.
Protein Preparations. Protein preparations of cSrc and cSrc
T338M were expressed and purified as described elsewhere.30 DNA
encoding wild-type human Abl (residues S229−Q513; isoform 1a
numbering) in a pET28a vector with an N-terminal His6-tag followed
by a thrombin cleavage site was purchased from GeneArt, Regensburg,
Germany. Abl protein was coexpressed with YopH (Yersinia outer
protein H) in Escherichia coli BL21-DE3 as described elsewhere.40
After harvest and cell lysis, Abl protein was enriched by Ni-NTA
chromatography, followed by gel filtration (High Load 16/60 Superdex
75, GE Healthcare) for buffer exchange (50 mM Tris, 150 mM
NaCl, 2.5 mM CaCl2, 0.1% v/v 2-mercaptoethanol, pH 8.0). Eluate
containing Abl protein was collected and the His-tag was removed by
digestion with thrombin (GE Healthcare) at 4 °C overnight. Cleaved
protein was isolated by ion exchange chromatography (MonoQ 5/50 GL;
GE Healthcare) using buffers A (50 mM Tris, 1 mM DTT, 5% v/v
glycerol, pH 8.5) and B (buffer A + 1 M NaCl) (0−30% B over 20 min;
1 mL/min). Finally, the buffer was adjusted to 25 mM Tris, 75 mM NaCl,
10% v/v glycerol, 5 mM DTT, pH 7.3, by gel filtration, and the protein
was flash-frozen in small aliquots. All purification steps were carried out at
4 °C. Correct mass was confirmed by ESI-MS. Abl T315I was purchased
from invitrogen (Lot no. 39639B, PV3866). KIT wild-type and KIT
V559D/T670I were purchased from Proqinase (Lot no. 010, 0997-0000-1
and Lot no. 002, 1044-0000-1)
Activity Based Assay for IC50 Determination. IC50 determi-
nations for cSrc, Abl, and KIT kinases were measured with the
KinEASE-TK assay from Cisbio according to the manufacturer’s
instructions. A biotinylated poly-Glu-Tyr substrate peptide was
phosphorylated by the specific kinase of interest. After completion
of the reaction, an anti-phosphotyrosine antibody labeled with
europium cryptate and streptavidin labeled with the fluorophore
XL665 were added. 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
wild-type cSrc, 1 μM for cSrc-T338M, 30 μM for wild-type Abl, 6 μM
for Abl T315I, 90 μM for wild-type KIT and 20 μM for KIT V559D/
T670I). A concentration of 100 nM substrate was used for wild-type
and drug-resistant cSrc as well as Abl, while 250 nM was used for Abl
T315I and 350 or 300 nM was used for KIT wild-type and KIT
V559D/T670I respectively. Kinase and inhibitor were preincubated
for 30−60 min before the reaction was started by addition of ATP and
substrate peptide. A Tecan Safire2 or Infinite M1000 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 determina-
tions of each IC50 were made.
Crystallization and Structure Determination of cSrc-3g and
cSrc-3h. For all complex structures, 2500 μM inhibitor (prepared in
DMSO) was preincubated along with 250 μM wild-type cSrc (stored in
20 mM Tris pH 8.0, 100 mM NaCl, 1 mM DTT) for 1 h on ice to form
the enzyme−inhibitor complex prior to crystallization. In the case of both
3g and 3h, crystals were grown using the hanging drop method at 20 °C
after mixing 1−1.5 μL of protein−inhibitor solution with 1 μL of reservoir
solution (125 mM MES (pH 6−7), 9−11.5% PEG 20000, 5% (v/v)
glycerol). All crystals were directly frozen without the addition of glycerol.
Diffraction data of the cSrc−3g complex crystal was measured in-house to
a resolution of 3.30 Å (Bruker AXS microstar); the other data set was
collected at the PX10SA beamline of the Swiss Light Source (PSI,
Villingen, Switzerland) to a resolution of 2.7 Å for cSrc−3h, using
wavelengths close to 1 Å. All data sets were processed with XDS41 and
scaled using XSCALE.41
Structure Determination and Refinement of cSrc−3g and
cSrc−3h. All cSrc−inhibitor complex structures were solved by mole-
cular replacement with PHASER42 using the published cSrc structure
2OIQ43 as template. The two cSrc molecules in the asymmetric unit
were manually modified using the program COOT.44 The model was
first refined with CNS45 using simulated annealing to remove model
bias. The final refinement was performed with REFMAC5.46 Inhibitor
topology files where generated using the Dundee PRODRG2 server.47
Refined structures were validated with PROCHECK.48 Data collection,
structure refinement statistics, PDB-ID codes, and further details for
the data collection, as well as Ramachandran plot results, are shown in
Table S1, Supporting Information. PyMOL49 was used to generate the
figures.
Reagents and Antibodies. Imatinib, sunitinib, dovitinib, and
ponatinib were purchased from LC Laboratories (Woburn, MA).
Rabbit polyclonal antibodies to KIT and phospho-KIT Y703 were
from DAKO (Carpinteria, CA) and Cell Signaling (Beverly Hills, CA),
respectively. Polyclonal rabbit antibodies to total p42/44 mitogen-
activated protein kinase (MAPK), phospho-p44/42 MAPK T202/
Y204, phospho-AKT S473, total AKT, were from Cell Signaling
(Beverly, MA). Beta Actin antibodies were purchased from Sigma
(St. Louis, MO).
Cellular Studies. GIST-T1, GIST-T1 T670I, and GIST-48B cell
lines were cultured as previously described.50 Viability studies were
carried out using a sulforhodamin (SRB) assay.51 For these studies, the
cell lines were plated at 15 000 to 30 000 cells per well in a 96-well flat-
bottom plate (Falcon, Lincoln, NJ), cultured in serum-containing
media for 1 day, and then incubated for 72 h with KIT inhibitors and
M
dx.doi.org/10.1021/jm4004076 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
DMSO-only solvent control. The SRB assay absorption was measured
with a Genion luminometer (Tecan, Crailsheim, Germany), and the
data were normalized to the DMSO-only control group. All
experimental points were measured in triplicate wells for each plate
and were replicated in at least two plates.
Western Blotting. Protein lysates were prepared from cell line
monolayers according to standard protocols.52 Protein concentrations
were determined with the Bio-Rad Protein Assay (Bio-Rad Laboratories,
Hercules, CA). Electrophoresis and immunoblotting were carried out as
previously described.53 Changes in protein expression and phosphor-
ylation as visualized by chemiluminescence were captured and quantified
using a FUJI LAS3000 system with Science Lab 2001 ImageGauge 4.0
software (Fujifilm Medial Systems, Stamford CT, USA).
GIST Cell Lines. GIST-T1 was established from a human,
untreated, metastatic GIST containing a 57 bp deletion in KIT exon
11, which is highly sensitive to imatinib at low nanomolar doses.35 GIST-
T1 harbors a homozygous deletion and is p53 negative as measured by
qRT-PCR and Western blot analysis. GIST-48 was established from a
GIST that had progressed, after initial clinical response, during imatinib
therapy. GIST-48 has a primary, homozygous KIT exon 11 missense
mutation (V560D) and a heterozygous secondary KIT exon 17 (kinase
activation loop) mutation (D820A).54 GIST-48 is imatinib-resistant, due
to a secondary KIT exon 17 mutation. GIST-48B is a subline of GIST-48,
which, despite retaining the activating KIT mutation in all cells, expresses
KIT transcript (data not shown) and protein at essentially undetectable
levels.55 GIST-48B is TP53 wild-type and expresses p53 RNA and protein
as measured by qRT-PCR and Western blot analysis. GIST-48 and GIST-
48B cell lines have been established at the Brigham and Women’s
Hospital in Boston, USA, by the Group of Professor Jonathan A. Fletcher.
GIST-T1 has been established by Takahiro Taguchi at the Kochi
University in Japan. GIST-T1 T670I was generated and kindly provided
by Prof. Brian Rubin, Department of Molecular Genetics, Lerner Research
Institute and Department of Anatomic Pathology and Taussing Cancer
Center, Cleveland Clinic, Cleveland, OH 44195.
cancer genomics-based drug discovery. RKT received consult-
ing and lecture fees (Sanofi-Aventis, Merck, Roche, Lilly,
Boehringer Ingelheim, Astra-Zeneca, Atlas-Biolabs, Daiichi-
Sankyo, Blackfield AG) as well as research support (Merck,
EOS and AstraZeneca). DR received consultant and lecture fees
from Astra-Zeneca, Merck-Serono, Takeda, Pfizer, Boehringer
Ingelheim and Sanofi-Aventis.
ACKNOWLEDGMENTS
■
We thank Brian Rubin for kindly providing GIST-T1 T670I
and Idoya Lahortiga for providing Ba/F3 cells. We thank
Jonathan A. Fletcher for providing the GIST48B and T.
Taguchi for providing the GIST T1. This work was cofunded
by the German federal state North Rhine Westphalia (NRW)
and the European Union (European Regional Development
Fund: Investing In Your Future), the German Federal Ministry
for Education and Research (NGFNPlus) (Grant Nos. BMBF
01GS08104 and 01GS08100), the EU-Framework Programme
CURELUNG (HEALTH-F2-2010-258677), the Deutsche
Forschungsgemeinschaft through TH1386/3-1 and through
SFB832, the Max Planck Society, the Behrens-Weise Founda-
tion (M.I.F.A.NEUR8061), Stand Up To Cancer−American
Association for Cancer Research Innovative Research Grant
(No. SU2C-AACR-IR60109), and an anonymous foundation.
This work was furthermore supported by funding from Max-
Eder Fellowship from the Deutsche Krebshilfe (S. Bauer) and
the Life Raft Group Research Initiative (S. Bauer). M.L.S. is a
fellow of the International Association for the Study of Lung
Cancer (IASLC).
ABBREVIATIONS
■
ASSOCIATED CONTENT
* Supporting Information
■
Abl Abelson kinase; Akt protein kinase B; Bcr breakpoint
cluster region; cSrc cellular sarcoma kinase; CYP cytochrome
P450; GIST gastrointestinal stromal tumors; KIT mast/stem
cell growth factor receptor kinase; LMS leiomyosarcoma; LPS
liposarcoma; MPNST malignant peripheral nerve sheath
tumor; GI50 concentration for 50% of maximal inhibition of
cell proliferation; IC50 concentration causing 50% inhibition of
enzyme activity; DMSO dimethyl sulfoxide; DIPEA N,N-
diisopropylethylamine; DCM dichloromethane; THF tetrahy-
drofuran; DMF dimethylformamide; NMP N-methylpyrrolidinone;
MES 2-(N-morpholino)ethanesulfonic acid; PEG poly(ethylene
glycol)
S
Detailed synthetic procedures for the preparation of 6a and 6b
as well as synthetic schemes for 3c,d, 4b, and 6a,b, crystal X-ray
structure data of 3g and 3h in complex with cSrc, modeling
studies of 3g into the catalytic domains of Abl T315I and KIT
T670I, selectivity profiling data of 3d, CYP inhibition data,
cytotoxicity data, Ba/F3 Bcr-Abl (and T315I) proliferation
assay data, and a detailed overview of 3h−cSrc interactions
including electron density information. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
PDB ID codes 3TZ7 and 3TZ8.
REFERENCES
■
AUTHOR INFORMATION
Corresponding Authors
*PD Dr. Sebastian Bauer: phone +49 (0)201-723 85558, fax
+49 (0)201-723 5547; e-mail sebastian.bauer@uk-essen.de.
*Prof. Dr. Daniel Rauh: phone +49 (0)231-755 7056, fax +49
(0)231-755 5159, e-mail daniel.rauh@tu-dortmund.de.
■
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