Synthesis and biological evaluation of 4-anilinoquinolines as potent inhibitors of epidermal growth factor receptor

Article abstract of DOI:10.1021/jm901877j

The mutant receptor tyrosine kinase EGFR is a validated and therapeutically amenable target for genotypically selected lung cancer patients. Here we present the synthesis and biological evaluation of a series of 6- and 7-substituted 4-anilinoquinolines as potent type I inhibitors of clinically relevant mutant variants of EGFR. Quinolines 3a and 3e were found to be highly active kinase inhibitors in biochemical assays and were further investigated for their biological effect on EGFR-dependent Ba/F3 cells and non-small cell lung cancer (NSCLC) cell lines.

Full text of DOI:10.1021/jm901877j

2892 J. Med. Chem. 2010, 53, 2892–2901
DOI: 10.1021/jm901877j
Synthesis and Biological Evaluation of 4-Anilinoquinolines as Potent Inhibitors of
Epidermal Growth Factor Receptor
Vijaykumar G. Pawar,† Martin L. Sos,‡ Haridas B. Rode,† Matthias Rabiller,† Stefanie Heynck,‡ Willem A. L. van Otterlo,†,
Roman K. Thomas,†,‡,§ and Daniel Rauh*,†
†Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany, ‡Max Planck Institute for
Neurological Research with Klaus-Joachim-Zu€lch Laboratories of the Max Planck Society and the Medical Faculty of the University of Ko€ln,
Gleueler Strasse 50, 50931 Ko€ln, Germany, §Department I of Internal Medicine and Center of Integrated Oncology Ko€ln;Bonn,
University of Ko€ln, 50924 Ko€ln, Germany, and Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand,
Johannesburg, South Africa
Received December 20, 2009
The mutant receptor tyrosine kinase EGFR is a validated and therapeutically amenable target for
genotypically selected lung cancer patients. Here we present the synthesis and biological evaluation of a
series of 6- and 7-substituted 4-anilinoquinolines as potent type I inhibitors of clinically relevant mutant
variants of EGFR. Quinolines 3a and 3e were found to be highly active kinase inhibitors in biochemical
assays and were further investigated for their biological effect on EGFR-dependent Ba/F3 cells and non-
small cell lung cancer (NSCLC) cell lines.
Introduction
compounds derived from ten-membered nitrogen-containing
bicyclic scaffolds. They concluded that the quinazoline core
was the best scaffold for the development of EGFR inhibitors
because any alteration of the nitrogen substitution pattern in
the bicyclic ring resulted in less active compounds.5 In parti-
cular, the replacement of the N3 of the quinazoline 1 by a CH
resulted in a 200-fold drop in affinity (Figure 1). At the same
time, based on modeling studies, scientists atWyeth6 hypothe-
sized that the quinazoline N3 interacts with the kinase domain
via a water-mediated hydrogen bond to the side chain of the
gatekeeper Thr790 of EGFR and provided a rationalization
for the crucial role of N3 of the quinazoline core for activity
(Figure 2a). On the basis of these findings, Wissner et al.7
developed a series of compoundswhere the N3 of the quinazo-
line was replaced by a C-CN group to: (a) displace the
hypothetical water molecule and (b) to serve as a hydrogen
bond acceptor for the Thr790 hydroxyl group (Figure 2b,c).
Through this elegant application of a bioisostere, Wissner and
colleagues were not only able to generate potent inhibitors of
EGFR (e.g. Neratinib, currently in clinical trials) but also to
resolve overlapping patent applications with Parke-Davis.
This binding mode of quinazolines to the hinge region of the
kinase domain and the interaction of N3 with the side chain of
the gatekeeper Thr790 viaawatermoleculewas coinedin2002
when researchers at Genentech published the crystal structure
of EGFR in complex with Erlotinib (PDB code: 1M17)8 and
has since then served as an important reference for the
structure-based design of both reversible and irreversible
EGFR and HER2 kinase inhibitors.7,9-11
The tyrosine kinase epidermal growth factor receptor
(EGFRa) and its closely related family member HER2 are
recognized targets for targeted cancer therapy and have been
extensively investigated by both academia and industry.1
Since the 1980s, when EGFR and HER2 were proposed as
potential anticancer targets, small molecule kinase inhibitors
and monoclonal antibodies have emerged as promising stra-
tegies to inhibit EGFR and HER2 kinase activity.2 To date,
the Food and Drug Administration has approved 11 small
molecule kinase inhibitors for targeted cancer therapy3 and
dozens are currently in clinical trials. Out of these 11 inhibi-
tors, Gefitinib, Erlotinib, and Lapatinib (Figure 1) belong to a
class of 4-anilinoquinazolines which have been designed to
target the ATP binding pocket of the kinase domain and are
approved for the treatment of EGFR/HER2-dependent tu-
mors which occur in non-small cell lung cancer (NSCLC) or
breast cancer.4 Although a plethora of quinazoline-based
kinase inhibitors are known and widely used in medicinal
chemistry and chemical biology research, examples of type I
kinase inhibitors based on the closely related quinoline scaf-
fold are few in number.
Type I inhibitors are ATP competitive and bind to the hinge
region of the enzymatically active kinase domain and repre-
sent the prototypical kinase inhibitor. One of the first exten-
sive SAR studies on type I inhibitors active on EGFR was
published in 1995 by researchers at Parke-Davis5 in which
Rewcastle and colleagues reported a SAR study on a series of
In our studies of the inhibition of EGFR and clinically
relevant drug resistant mutant variants by reversible and
irreversible 4-anilinoquinazolines,12 we could not correlate
the importance of the discussed water-mediated hydrogen
bond with potent inhibition of EGFR. This stimulated our
interest in designing and synthesizing of a series of 6,7-
substituted 4-anilinoquinolines to compare SAR with their
*To whom correspondence should be addressed. Phone: þ49 (0)231-
9742 6480. Fax: þ49 (0)231-9742 6479. E-mail: daniel.rauh@cgc.mpg.
de.
aAbbreviations: EGFR, epidermal growth factor receptor; TKIs,
tyrosine kinase inhibitors; HTRF, homogeneous time-resolved fluore-
scence; NSCLC, non-small cell lung cancer; GI50, concentration at
which 50% growth retardation is observed.
r
pubs.acs.org/jmc
Published on Web 03/11/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2893
Figure 1. Structures of 4-anilinoquinazoline- and 4-anilinoquinoline-based EGFR/HER2 inhibitors. Simple 4-anilinoquinazolines such as 1
served as starting points for the development of potent type I and type II EGFR/HER2 inhibitors approved for the treatment of NSCLC.
Erlotinib and Gefitinib are type I inhibitors and bind to the ATP site of activated kinases. Lapatinib is a type II inhibitor and stabilizes
enzymatically inactive kinase conformations of EGFR and HER2. According to earlier studies, quinolines such as 2a show reduced activity
against EGFR and are less suited for compound optimization.5 2b represents a series of 6- and 7- substituted 4-anilinoquinolines as potent type
I inhibitors of clinically relevant mutant variants of EGFR discussed in this study.
Figure 2. Binding modes of 4-anilinoquinazoline- and 4-anilinoisoquinoline-based EGFR and HER2 inhibitors. (a) Proposed binding mode
of a 4-anilinoquinazoline to the ATP-binding site of EGFR. Hydrogen bonding interactions of the inhibitor with the hinge region (pink) and
the side chain of the gatekeeper residue Thr790 (mediated via a water molecule (W)) are shown as red dotted lines. (b) This binding mode
stimulated the design and synthesis of 3-quinolinecarbonitriles to displace the proposed water molecule and to form a direct hydrogen bond to
the side chain of gatekeeper residue (Thr790).7 (c) The presence of a less conserved Cys at the lip of the ATP pocket fostered the development of
irreversible quinazoline and isoquinoline EGFR inhibitors currently in clinical trials. The irreversible inhibitor Neratinib is modeled to the
catalytic domain of EGFR. Modeling is based on the crystal structure of Neratinib in complex with drug resistant EGFR-Thr790Met (PDB
code: 2JIV).15 The electrophile of the inhibitor forms a covalent bond with the side chain of Cys797. Cys797 is located at the N-terminal end of a
short helix and activated by the helix dipole. Both reversible and irreversible 3-quinolinecarbonitriles were designed to (a) displace the water
molecule (W10), and (b) to form a direct hydrogen bond to the side chain hydroxyl of the gatekeeper Thr790.7
direct quinazoline-based homologues and to investigate their
inhibition of EGFR and its clinically relevant mutant var-
iants, both in biochemical and in cellular assays.
the platelet-derived growth factor receptor-R (PDGFRR,
Thr674Ile), and EGFR (Thr790Met). All of them result in
tumor drug resistance.13,14 In wild type kinases, the gate-
keeper residue is a smaller amino acid located in the hinge
region of the kinase domain and controls access to the
hydrophobic subpocket, thereby playing a crucial role in
inhibitor affinity and selectivity (Figure 2).2 It has also been
shown that these mutations prevent binding of kinase inhibi-
tors such as Imatinib, Gefitinib, and Erlotinib due to steric
hindrance and/or an increase in ATP affinity,15 highlighting
the necessity for the development of novel strategies to design
inhibitors capable of overcoming mutation-associated drug
resistance in kinases.16 Our previous structural and functional
Results and Discussion
The design and synthesis of the quinoline inhibitors evolved
from our previous studies aimed to address questions of how
to overcome Thr790Met drug resistance in EGFR using
small organic molecules.12 One of the key challenges in
targeted cancer therapy is the emergence of drug resistance
mutations in the primary target. At the gatekeeper position,
mutations leading to sterically demanding amino acids have
been observed in BCR/ABL (Thr315Ile), c-KIT (Thr670Ile),

2894 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Pawar et al.
Table 1. Focused Library of Designed 4-Anilinoquinoline-Based Inhibitors and their 4-Anilinoquinazoline Counterpartsa
a The structures of reversible and irreversible 4-anilinoquinoline- and 4-anilinoquinazoline-based inhibitors are shown. On the basis of their binding
mode to the kinase domain of EGFR, the individual inhibitors are classified as follows: irreversible 3a; reversible 3c, 3e, and 4a,b, quinolines and
irreversible 3b; reversible 3d, 3f, Erlotinib and 4c, quinazolines. IC50 values were measured with an activity based phosphorylation assay (HTRF). Both
reversible and irreversible quinolines give IC50 values more or less comparable to their quinazoline counterparts, demonstrating that the 4-
anilinoquinoline-core is indeed suitable for targeting EGFR.
studies alluded toward the possibility of a steric clash between
the Met gatekeeper in drug resistant EGFR-Thr790Met and
the 3-bromoanilino moiety at the 4-position of quinazoline
inhibitors.12,17 Wereasoned thatthe bindingof ligandssuch as
erlotinib to EGFR-Thr790Met would not only be hampered
by this steric hindrance but also due to the loss of the water-
mediated hydrogen bond to the side chain hydroxyl of the
Thr790 of EGFR-WT, an interaction which is not possible
with the side chain of the mutated Met790. However, we were
puzzled by the fact that although EGFR-Thr790Met is a drug
resistant mutation with fatal outcome in affected patients,
reversible quinazolines such as Erlotinib and Gefitinib lose
potency by only 2-3 orders of magnitude while irreversible
4-anilinoquinazolines still inhibit EGFR-Thr790Met in the
subnanomolar range in biochemical assays.12,17 We hypothe-
sized that if the N3 of the quinazoline does play an essential
role in binding to EGFR, via the formation of a hydrogen
bond, the gatekeeper Thr to Met mutation should have
resulted in a much more dramatic loss of activity. Therefore,
we investigated the requirement of a nitrogen atom at the 3-
position of the ten-membered ring system (e.g., 2a, Figure 1)
for potent inhibition of EGFR and proposed, contrary to the
SAR reported by Rewcastle et al.,5 that 6,7-substituted qui-
nolines (2b, Figure 1) would also be effective inhibitors of
EGFR. Excluding the direct steric effect of the Met790 side
chain on binding, we believed that the binding mode of
the quinoline inhibitor (2b, Figure 1) to EGFR-WT would
be comparable to that of an analogous quinazoline-based

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2895
Scheme 1. Synthesis of Quinoline and Quinazoline Derivatives Ia
a Reagents and conditions: (a) HNO3/H2SO4, -15 to 0 °C; (b) POCl3, cat. DMF, reflux; (c) 3-bromoaniline, cat. conc HCl, isopropyl alcohol, reflux;
(d) DMF acetal, reflux; (e) 3-bromoaniline, HOAc, reflux; (f) Fe, HOAc, aq EtOH, reflux; (g) RCOCl, DIEA, THF, 0 °C to rt; (h) oxalyl chloride,
CH2Cl2, cat. DMF, reflux, 30 min. Compound 3f was synthesized from 11b and 4-(dimethylamino)butyric acid hydrochloride 12, EDCI.HCl,
triethylamine, DMF, 0 °C to rt, overnight.
Scheme 2. Synthesis of Quinoline and Quinazoline Derivatives IIa
a Reagents and conditions: (a) POCl3, cat. DMF, reflux; (b) 3-ethynylphenylamine, cat. conc HCl, isopropyl alcohol, reflux.
inhibitor bound to drug resistant EGFR-Thr790Met because
hydrogen bonding with a Thr790 is not possible in either
situation. However, taking these direct steric effects into
consideration, the potency of the 4-anilinoquinoline inhibi-
tors against EGFR-WT would be expected to be better than
the potency of the more traditional quinazoline-based inhibi-
tors like Erlotinib against EGFR-Thr790Met.
Interestingly, at the time we were synthesizing a series of 4-
anilinoquinolines to test our hypotheses, the structure factors
of the EGFR-Erlotinib complex were made available to the
public (PDB code: 1M17).8 This allowed us to calculate the
corresponding electron density maps (Supporting Informa-
tion Figure 1), and we were surprised to find that there was no
electron density supporting the existence of a water molecule
mediating the binding of N3 of the quinazoline core to the side
chain of Thr790, further stimulating our design of quinoline-
based type I kinase inhibitors. To explore the SAR, based on
our prediction that the N3 of the quinazoline core should not
Figure 3. Proposed binding mode of 4-anilinoquinolines to EGFR.
play a central role in binding to EGFR, we synthesized
different quinoline and quinazoline inhibitors and screened
these against EGFR and its mutant variants (Leu858Arg and
Leu858Arg-Thr790Met) in biochemical (Table 1) and cellular
assays (Figure 4), respectively.
Chemistry. Quinoline and quinazoline inhibitors (3a-f
and 4a-c, Table 1) were designed to perform comparative
SAR studies. 4-Anilinoquinolines 3a,c,e (Table 1) and 4-
anilinoquinazolines 3b,d,f (Table 1) were synthesized start-
ing from the intermediates 8a and 8b, respectively. These
intermediates were synthesized by two different routes as
described in Scheme 1. Synthesis of 8a was achieved by
regioselective nitration of 4-hydroxyquinoline 5 at the 6-
position, using nitric acid in sulfuric acid at -15 °C, to afford
(a) 3e is modeled into the ATP-binding site of EGFR based on the
crystal structure of Erlotinib in complex with EGFR (PDB code:
1M17) and an irreversible quinazoline in complex with cSrc (PDB
code: 2QLQ). The ring nitrogen of the quinoline forms a hydrogen
bond (red dotted line) with the backbone of the hinge region (pink).
The protonated tertiary amine of 3e is within hydrogen bonding
distance to the side chain of Asp800. This additional interaction is
not observed for different substituted quinolines and helps to
explain the gain in affinity for 3e. (b) The irreversible 4-anilinoqui-
noline 3a is modeled into the ATP-binding site of EGFR based on
the crystal structure of its quinazoline counterpart to EGFR (PDB
code: 2J5F).26 The electrophile of the inhibitor forms a covalent
bond to the side chain of Cys797.
6-nitro-4-hydroxyquinoline 6. Chlorination of 6 resulted in
the 4-chloro-6-nitroquinoline 7, which was heated at reflux

2896 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Pawar et al.
Figure 4. 4-Anilinoquinolines inhibit growth of EGFR-dependent cancer cells. (a) GI50 values for 4-anilinoquinoline- and 4-anilinoquinazo-
line-based EGFR inhibitors screened against Ba/F3 cell lines expressing different EGFR mutants with or without the secondary Thr790Met
gatekeeper mutation (TM). The range of compound activity is color-coded. (b) The EGFR mutant NSCLC cell line PC9 was treated with
increasing concentrations of 3a (irreversible) and 3e (reversible), and cell viability was determined using measurement of ATP content. The
error bars represent the standard deviation for three replicates. (c) The NSCLC cell line H1975 expressing the Thr790Met EGFR gatekeeper
mutation was treated with increasing concentrations of 3a and 3e, and cell viability was determined using measurement of ATP content. The
error bars represent the standard deviation for three replicates. (d) Inhibition of the activation status of EGFR was determined by
immunoblotting after treatment of PC9 cells with increasing concentrations of 3a and 3e (from left to right: control, 0.1, 0.3, 1, 3, 10 μM);
actin was detected as loading control.
with 4-bromoaniline and catalytic amounts of hydrochloric
acid in isopropyl alcohol to give 8a. The intermediate 8b,
which was required for the synthesis of quinazolines 3b, 3d,
and 3f, was obtained by starting from 2-amino-5-nitroben-
zonitrile 9, which on treatment with dimethylformamide-
dimethylacetal (DMF-DMA) in toluene produced the N,N-
dimethylformamidine derivative 10 (Scheme 1). Com-
pound 10 was heated at reflux with 3-bromoaniline in acetic
acid to give intermediate 8b. Finally, the nitro groups of 8a,b
were reduced using iron in acetic acid, followed by acylation
of the resulting amines 11a,b with the corresponding acid
chlorides to obtain the 4-anilinoquinoline and quinazoline
derivatives 3a-e (Scheme 1). 4-(Dimethylamino)butanoyl
chloride 13 required for 3e was synthesized in situ from the
corresponding acid 12 using oxalyl chloride and catalytic
amounts of DMF. Compound 3f was synthesized from 11b
and 4-(dimethylamino)butyric acid hydrochloride 12, using
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochlor-
quinazolines 4a-c, were prepared by using procedures de-
scribed in the literature.17-19 The 4-hydroxyquinoline and
quinazoline derivatives 14a-c were subsequently treated
with phosphorus oxychloride and catalytic N,N0-dimethyl-
formamide to afford 15a-c. Finally, 4a-c were obtained
from 15a-c by the treatment with commercially available 3-
ethynylphenylamine and a catalytic amount of concentrated
hydrochloric acid in isopropyl alcohol (Scheme 2).
Biochemical Screening. To test the above-mentioned hy-
potheses regarding inhibition of EGFR by 4-anilinoquino-
lines, enzyme activity assays were performed for EGFR wild
type and the two clinically relevant mutant variants EGFR-
Leu858Arg (inhibitor-sensitizing mutation) and EGFR-
Leu858Arg-Thr790Met (drug resistant mutation), respec-
tively (Table 1). Although potencies of the inhibitors varied
between the tested mutant kinases, the measured IC50 values
showed the expected trends for reversible and irreversible
inhibitors. We observed that all reversible and irreversible
4-anilinoquinoline-based inhibitors showed low to sub-
nanomolar IC50 values on EGFR wild type and EGFR-
Leu858Arg. When compared to their reversible quinazoline
ide (EDCI HCl) as a coupling reagent. The 4-hydroxyquino-
3
line and quinazoline derivatives 14a-c, required for the
synthesis of the 6,7-disubstituted 4-anilinoquinolines and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2897
Figure 5. Kinase selectivity profile of 3e and 3f. Kinase profiling was performed for 3e and 3f against a panel of 95 different protein kinases at a
concentration of 10 μM (National Centre for Protein Kinase Profiling, University of Dundee, UK). The screening was carried out using a
radioactive (33P-ATP) filter-binding assay. The scores represent the % activity remaining relative to the 100% DMSO controls. The inhibition
is color coded from red (strong inhibition, low score) to green (weak inhibition, high score). Both quinazoline- and quinoline-based inhibitors
show preference for kinases with a small amino acid at the gatekeeper position (Thr).
counterparts, quinolines 3c, 4a (corresponding quinoline to
the marketed drug Erlotinib), and 4b showed decreased
potency by 1-2 orders of magnitude. Interestingly, this trend
was not observed for 3e, a quinoline containing an N,N-
dimethylamino-butynoyl group in the 6-position of the
quinoline core. The design of this compound and its quinazo-
line counterpart 3f was stimulated by a charged interaction
we previously observed for irreversible quinazoline-based
EGFR inhibitors, decorated with an (E)-4-(dimethylamino)-
but-2-enamide electrophile in the quinoline 6-position,
bound to mutant variants of the tyrosine kinase cSrc (PDB
codes: 2QQ7 and 2QLQ).12 In these structures, the N,N-
dimethylamino group is within hydrogen bonding distance
to the side chain of Asp348. cSrc and EGFR share high
identity in sequence (37% overall) and structure and the ATP
binding sites are highly conserved (70%). Structural model-
ing of the identified binding mode in cSrc to the kinase
domain of EGFR supported that this interaction of the
protonated tertiary amine of the inhibitor with the side
chain of Asp348 in cSrc could be conserved in EGFR via
the isostructural Asp800 and should result in increased
potency when introduced into reversible inhibitors (Figure 3A).
While all reversible quinolines showed a decreased affinity in
drug resistant EGFR-Leu858Arg-Thr790Met, the irreversible
inhibitors 3a and 3b retained potency in the subnanomolar
range. These trends are well documented for 4-aminoquinazo-
lines,12 but to the best of our knowledge have not been
reported for 4-aminoquinolines and can be directly linked to
the enlarged amino acid side chain at the gatekeeper position.
Profiling the Cellular Activity of Quinolines. To determine
the activity of the 4-anilinoquinolines in vitro, cellular-
based screening of all the synthesized quinolines as well as
on the structurally corresponding quinazolines and Erloti-
nib was performed in EGFR-dependent Ba/F3 cell lines.
These cell lines express a series of clinically relevant EGFR-
mutations, either with or without the Thr790Met gate-
keeper mutation in EGFR (Figure 4a).12 We found the
quinolines to be active in all EGFR mutant Ba/F3 cells that

2898 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Pawar et al.
did not express the gatekeeper mutation, with GI50 values
within the range of their structurally corresponding quina-
zolines. We next tested the ability of quinoline 3a and 3e to
inhibit cell growth of patient derived NSCLC cell lines
expressing mutant EGFR. Recapitulating our previous
results, both quinolines robustly decreased viability of
EGFR-mutated PC9 cells at submicromolar concentra-
tions in the range of the activity of the quinazoline Erlotinib
(Figure 4b). As expected, the irreversible quinoline 3a
is also active in cells (H1975) expressing the gatekeeper
mutation Thr790Met in EGFR (Figure 4b). To assess the
on-target activity of the quinolines, we determined the
phosphorylation status of EGFR in PC9 cells after treat-
ment with either 3a or 3e (Figure 4c), and this showed
inhibition of EGFR autophosphorylation in a dose depen-
dent manner.
Kinase Profiling. To compare the kinase inhibitor selecti-
vity of our newly developed 4-anilinoquinolines with their
quinazoline counterparts, kinase profiling was performed
for 3e and 3f against a panel of 95 different protein kinases
measuring the inhibition of kinase activity at a concentration
of 10 μM (National Centre for Protein Kinase Profiling,
University of Dundee, UK) (Figure 5). Both compounds
showed the highest inhibitory effect, with equal potency, on
the receptor tyrosine kinase HER4, the tyrosine kinases Lck
and Yes1, as well as on the serine/threonine kinase RIPK2.
Of particular interest was the finding that all these kinases
possess a threonine at the gatekeeper position, suggesting
that the N3 of the quinazoline inhibitor 3f is unlikely to play
a significant role in binding to these kinases. For the kinases
that are weakly or moderately inhibited, a preference for one
inhibitor over the other was observed. For example, the
potency of the quinoline 3e was higher than for the quinazo-
line counterpart 3f, against EPBH-3, cSrc (Thr), and
MINK1 (Met), while the quinazoline 3f exhibited higher
activity against PHK (Phe), PIM1 (Leu), and SmMLCK
(Leu) kinases.
Experimental Section
Chemistry. General. Unless otherwise noted, all reagents and
solvents were purchased from Acros, Fluka, Sigma Aldrich, or
Merck and used without further purification. 1H and 13C NMR
spectra were recorded on a Bruker Avance DRX 400 spectro-
meter at 400 and 125 MHz or 100 MHz, respectively. Chemical
shifts (δ) are given in ppm downfield from tetramethylsilane as
an internal standard. 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 mon-
itored by UV at 254 nm. 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).
All tested compounds possessed a purity of >95% as deter-
mined by HPLC coupled with mass spectroscopy (HPLC-ESI-
MS). HPLC-ESI-MS analyses were performed on an HPLC
system from Agilent (1200 series) with an Eclipse XDB-C18,
5 μm (column dimensions: 150 mm ꢀ 4.60 mm) column from
Agilent and UV detection at 254 nm coupled to a Thermo
Finnigan LCQ Advantage Max ESI-spectrometer. H2O with
0.1% formic acid (solvent A) and acetonitrile with 0.1% formic
acid (solvent B) was used at a flow of 1 mL/min as a solvent
system for HPLC-ESI-MS along with the gradient: 0 min, 90%
solvent A/10% solvent B; 1 min, 90% solvent A/10% solvent B;
10 min, 0% solvent A/100% solvent B; 12 min, 0% solvent A/
100% solvent B; 15 min, 90% solvent A/10% solvent B.
N-(4-(3-Bromophenylamino)quinolin-6-yl)acrylamide (3a). Com-
pound 3a was prepared according to the literature procedure12
using N4-(3-bromophenyl)quinoline-4,6-diamine 11a (0.32 g,
1.02 mmol), 250 mM solution of acryloyl chloride (4.07 mL,
1.02 mmol), N,N-diisopropylethylamine (266 μL, 1.5 mmol), and
THF (103 mL) to produce the 62.0 mg (16.5%) of white solid after
the preparative HPLC purification. 1H NMR (400 MHz, DMSO-
d6) δ 10.85-10.42 (m, 2H), 9.07 (s, 1H), 8.50 (d, J = 6.8 Hz, 1H),
8.11-7.92 (m, 2H), 7.70 (s, 1H), 7.61-7.55 (m, 1H), 7.62-7.47 (m,
2H), 6.92 (d, J= 6.8 Hz, 1H), 6.53 (dd, J= 10.1, 17.0 Hz, 1H), 6.35
(dd, J = 1.9, 17.0 Hz, 1H), 5.86 (dd, J = 1.9, 10.1 Hz, 1H). 13
C
Conclusion
NMR (125 MHz, DMSO) δ 164.47, 155.02, 142.95, 140.37, 138.52,
136.04, 132.59, 132.17, 130.59, 128.86, 128.62, 128.49, 124.82,
123.11, 122.36, 119.02, 112.59, 101.58. HRMS (ESI-MS) calcd
368.0393 for C18H15BrN3O [M þ Hþ], found 368.0397. LC-MS
The improved understanding of kinase biology in disease
states and a more precise description of inhibitor scaffold
interactions with their target proteins has resulted in the
design and development of a steadily increasing arsenal of
potent and selective kinase inhibitors for targeted therapies
and chemical biology research. Among the classical kinase
inhibitors, compounds derived from ten-membered nitro-
gen-containing bicyclic scaffolds such as quinazolines repre-
sent a privileged chemotype that forms hydrogen bonds to
the hinge region of the kinase domain and mimics the
adenine ring of ATP. Several potent, quinoline-based type
II inhibitors, which not only bind to the hinge region of the
ATP pocket but also extend past the gatekeeper residue into
an adjacent allosteric site to stabilize enzymatically inactive
DFG-out kinase conformations, have been reported for
p38R20 and VEGFR,21 respectively. However, quinoline-
based type I inhibitors have been presumed to be suboptimal
and are therefore clearly underrepresented in kinase inhibi-
tor literature. In the present study, we demonstrate that
reversible as well as irreversible inhibitors based on the 4-
anilinoquinoline scaffold can be as potent as their quinazo-
line counterparts. In light of this observation, it is thus felt
that they may serve as valuable starting points for further
compound design.
tR = 6.7 min, purity g 99%.
N-(4-(3-Bromophenylamino)quinolin-6-yl)propionamide (3c).
A 10 mL round-bottom flask was flushed with argon and
charged with 11a (0.1 g, 0.32 mmol), DIEPA (0.14 mL, 0.67
mmol), and dry THF and cooled down to 0 °C. Then a 100 mM
solution of propionylchloride (0.28 mL, 0.32 mmol) was added
dropwise to the reaction mixture. The reaction was stirred for an
hour from 0 °C to room temperature. The progress of reaction
was monitored by TLC, and upon completion the reaction
mixture was basified with 10% aq NaHCO3. The water phase
was extracted with ethyl acetate (3 ꢀ 10 mL), and the combined
organic layers were washed with water and dried over Na2SO4.
The volatiles were removed in vacuo affording a yellow solid,
which was further purified by column chromatography using
DCM/MeOH (8:1) as an eluent to produce 42 mg (35%) of the
title compound. 1H NMR (400 MHz, DMSO-d6) δ 10.17 (s, br,
1H), 9.02 (s, br, 1H), 8.62 (d, J = 2 Hz, 1H), 8.46 (d, J = 5.1 Hz,
1H), 7.86 (d, J = 9.0 Hz, 1H), 7.76 (dd, J = 2, 9.0 Hz, 1H), 7.50
(s, 1H), 7.38-7.28 (m, 2H), 7.22 (dd, J = 3.4, 5.4 Hz, 1H), 7.08
(d, J = 5.1 Hz, 1H), 2.40 (q, J = 7.5 Hz, 2H), 1.13 (t, J = 7.5,
3H). 13C NMR (100 MHz, DMSO-d6) δ 172.99, 149.76, 147.27,
146.43, 144.44, 137.01, 132.02, 130.24, 125.69, 124.50, 123.54,
122.95, 121.89, 119.85, 111.26, 105.15, 30.34, 10.53. HRMS

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2899
(ESI-MS) calcd 370.0550 for C18H17BrN3O [M þ Hþ], found:
370.0553. tR = 6.5 min, purity = 98%.
(s, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.52-7.37 (m, 2H), 7.26 (s,
1H), 4.07 (s, 3H), 4.08 (s, 3H), 3.62 (s, 1H). 13C NMR (100 MHz,
MeOD) δ 159.06, 158.05, 151.85, 148.45, 137.34, 135.83, 130.15,
129.16, 127.82, 124.92, 123.48, 107.80, 102.74, 99.34, 82.59,
78.60, 56.34, 56.22. HRMS (ESI-MS) calcd 306.1237 for C19H16-
N3O2 [M þ Hþ], found 306.1236. tR = 6.15 min, purity = 97.4%.
N-(4-(3-Bromophenylamino)quinazoline-6-yl)acrylamide (3b).
The compound is prepared according to the literature proce-
dure12 from N4-(3-bromophenyl)-4,6-quinazolinediamine (0.3 g,
0.95 mmol), acryloyl chloride (95 μL, 1.14 mmol), N,N-diiso-
propylethylamine (380 mL, 2.29 mmol), and THF (103 mL) to
produce 40 mg (11%) of white solid upon purification using
N-(4-(3-Bromophenylamino)quinolin-6-yl)-4-(dimethylamino)-
butanamide (3e). (E)-4-(Dimethylamino)but-2-enoic acid hydro-
chloride (80 mg, 0.32 mmol) was stirred in 4 mL of THF under
argon for 5 min. The solution was then cooled in an ice bath and
DMF (50 μL) was added, followed by dropwise addition of oxalyl
chloride (85 μL, 1.0 mmol). The reaction was stirred at room
temperature for 2 h until a deep-orange color was observed. Then
the reaction mixture was cooled in an ice bath for 10 min, and a
solution of 11a (0.1 g, 0.32 mmol) in 1-methyl-2-pyrrolidinone was
added dropwise. Stirring was continued for the next 30 min in an
ice bath, followed by stirring at room temperature for 2 h. Upon
completion of the reaction, a saturated aqueous solution of
NaHCO3 was added to the reaction mixture until it became
alkaline (pH 8-9). The resulting solid was then filtered off and
purified by column chromatography (5-10% MeOH in DCM) to
obtain a yellow solid product 3e (56 mg, 41%); mp 186-187 °C. 1H
NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 8.91 (s, 1H), 8.58 (s,
1H), 8.44 (d, J = 4.9 Hz, 1H), 7.83 (d, J = 9.0 Hz, 1H), 7.73 (dd,
J = 2.2, 9.1 Hz, 1H), 7.47 (s, 1H), 7.33-7.26 (m, 3H), 7.18 (d, J =
6.9 Hz, 1H), 7.06 (d, J = 4.9 Hz, 1H), 2.38 (t, J = 7.3 Hz, 2H), 2.25
(t, J = 7.3 Hz, 2H), 2.13 (s, 6H), 1.74 (qn, J = 7.3 Hz, 2H). 13C
NMR (100 MHz, DMSO-d6) δ 172.30, 147.16, 146.63, 144.35,
136.91, 132.04, 130.41, 125.73, 124.42, 123.56, 122.95, 121.85,
119.88, 111.18, 105.13, 59.27, 45.90, 34.98, 23.78. HRMS (ESI-
MS) calcd 427.1128 for C21H24BrN4O [M þ Hþ], found 427.1126.
tR = 5.19 min, purity = 99.0%.
1
preparative HPLC. H NMR (400 MHz, DMSO-d6): δ 10.48
(bs, 1H), 9.91 (bs, 1H), 8.82 (d, J = 2.2 Hz, 1H), 8.59 (s, 1H),
8.19 (t, J = 1.9 Hz, 1H), 7.93-7.85 (m, 2H), 7.79 (d, J = 9.0 Hz,
1H), 7.34 (t, J = 8.0 Hz, 1H), 7.31-7.26 (m, 1H), 6.53 (dd, J =
10.1, 17.0 Hz, 1H), 6.35 (dd, J = 1.9, 17.0 Hz, 1H), 5.83 (dd, J =
1.9, 10.1 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): δ 163.34,
157.28, 153.18, 146.83, 141.14, 136.63, 131.54, 130.33, 128.56,
127.48, 127.24, 125.89, 124.24, 121.20, 120.77, 115.49, 112.24.
HRMS (ESI-MS): calcd for C17H14BrN4O [M þ Hþ] 369.03455,
found 369.03506. tR = 7.12 min, purity = 99.4%.
N-(4-(3-Bromophenylamino)quinazoline-6-yl)propionamide (3d).
The compound was synthesized from N4-(3-bromophenyl)-4,6-
quinazolinediamine (0.1 g, 0.32 mmol), propionyl chloride
(31 μL, 0.47 mmol), and N,N-diisopropylethylamine (110 μL,
0.67 mmol) in 3.5 mL of THF according to the literature.22 An
off-white solid (40 mg, 37%) was produced upon preparative
HPLC. 1H NMR (400 MHz, DMSO-d6): δ 10.2 (s, 1H), 9.90 (s,
1H), 8.73 (s, 1H), 8.57 (s, 1H), 8.17 (s, 1H), 7.89-7.81 (m, 2H),
7.77 (d, J = 8.9 Hz, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.28 (d, J =
8.0 Hz, 1H), 2.41 (q, J = 7.5 Hz, 2H), 1.14 (t, J = 7.5 Hz, 3H).
13C NMR (125 MHz, DMSO-d6): δ 172.13, 157.22, 152.86,
146.46, 141.21, 137.05, 130.26, 128.36, 127.16, 125.77, 124.23,
121.09, 120.81, 115.45, 111.62, 29.36, 9.56. HRMS (ESI-MS):
N-(3-Ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinolin-4-amine
(4a). 3-Ethynylaniline 37.6 mg (0.32 mmol) and catalytic
amount of 2N HCl (50 μL) was added to the solution of 4-
chloro-6,7-bis(2-methoxyethoxy)quinoline (0.1 g, 0.32 mmol) in
isopropyl alcohol, and reaction mixture was heated under reflux
for 2 h. The reaction mixture was cooled to room temperature
and basified with 10% aq sodium bicarbonate, followed by
extraction with CHCl3/MeOH (4:1). The combined organic
layers were washed with water and dried over Na2SO4. The
volatiles were removed in vacuo and resulting material was
purified with column chromatography using CH2Cl2/MeOH
(100:1 to 100:7) as an eluent to afford a 42 mg (33%) of yellow
solid . 1H NMR (400 MHz, CD3CN) δ 9.52 (s, 1H), 8.06 (d, J =
6.8 Hz, 1H), 7.60 (s, 1H), 7.56-7.42 (m, 5H), 6.76 (d, J = 6.8 Hz,
1H), 4.31 (dd, J = 3.7, 5.3 Hz, 2H), 4.26-4.21 (m, 2H),
calcd for C17H1679BrN4O [M
371.05074. tR = 8.71 min, purity = 98.2%.
þ
Hþ] 371.05020, found
N-{4-[(3-Bromo-phenyl)amino]-quinazoline-6-yl}-4-(dimethyl-
amino)butanamide (3f). 4-(Dimethyl amino)-butyric acid hydro-
chloride (0.1 g, 0.506 mmol) and triethylamine (0.18 mL,
2.02 mmol) were stirred in 3 mL of DMF at 0 °C for 10 min.
EDC HCl (0.130 g, 1.01 mmol) was added, and the reaction
3
mixture was further stirred for 10 min. The N4-(3-bromo-
phenyl)-4,6-quinazolinediamine (0.1 g, 0.31 mmol) was then
added as a solid, and the reaction mixture was stirred at 0 °C for
30 min and then overnight at room temperature. On the next
day, 0.5 mL of triethylamine was added, and the reaction
mixture was extracted with ethyl acetate, washed with brine,
and the organic phase was dried with sodium sulfate. Ethyl
acetate was removed to obtain an oily residue which was
purified by preparative HPLC to obtain 27 mg (19%) of a
pale-yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 10.25 (s,
1H), 9.88 (s, 1H), 8.72 (d, J = 1.9 Hz, 1H), 8.57 (s, 1H), 8.16 (t,
J = 1.9 Hz, 1H), 7.89-7.81 (m, 2H), 7.75 (d, J = 8.9 Hz, 1H),
7.34 (t, J = 8.0 Hz, 1H), 7.30-7.26 (m, 1H), 2.42 (t, J = 7.4 Hz,
2H), 2.27 (t, J = 7.1 Hz, 2H), 2.15 (s, 6H), 1.77 (qn, J = 7.2 Hz,
2H). 13C NMR (125 MHz, DMSO-d6): δ 171.36, 157.23, 152.86,
146.48, 141.21, 137.05, 130.25, 128.37, 127.13, 125.77, 124.25,
121.07, 120.83, 115.45, 111.53, 58.46, 45.11(2 ꢀ C), 34.02, 22.92.
HRMS (ESI-MS): calcd for C20H23BrN5O [M þ Hþ] 428.10805,
found 428.10792. tR = 6.18 min, purity = 98.3%.
3.83-3.75 (m, 4H), 3.50 (s, 1H), 3.43 (s, 3H), 3.41 (s, 3H). 13
C
NMR (100 MHz, CD3CN) δ 154.76, 153.21, 149.54, 139.92,
138.35, 136.04, 130.62, 130.56, 128.13, 125.73, 123.91, 112.40,
102.59, 101.28, 99.99, 82.63, 79.51, 70.57, 70.38, 69.15, 69.02,
58.64, 58.62. HRMS (ESI-MS) calcd 393.1809 for C23H25N2O4
[M þ Hþ], found 393.1812. tR = 6.56 min, purity = 98.7%.
N-(3-Ethynylphenyl)-6,7-dimethoxyquinolin-4-amine (4b). Com-
pound 4b was synthesized in the similar way as described for 4a
using 4-chloro-6,7-dimethoxyquinoline (0.2 g, 0.9 mmol), 3-ethynyl-
aniline (0.1 g, 0.9 mmol), and catalytic amount of 2N HCl (50 μL).
The crude was purified over silica gel using CH2Cl2/MeOH (100:1 to
100:5) as an eluent affording 0.24 g (86%) of 4b;mp253-255 °C. 1H
NMR (400 MHz, MeOD) δ 8.25 (d, J = 7.0 Hz, 1H), 7.88 (s, 1H),
7.60-7.48 (m, 4H), 7.30 (s, 1H), 6.86 (d, J = 7.0 Hz 1H), 4.10-4.00
(m, 6H), 3.68 (s, 1H). 13C NMR (100 MHz, Me3OD) δ 156.29,
154.32, 150.96, 139.61, 138.18, 136.05, 131.02, 130.41, 128.77,
126.02, 124.74, 112.39, 101.52, 99.66, 99.58, 82.19, 79.33, 56.08,
56.06. HRMS (ESI-MS) calcd 305.1285 for C19H17N3O2 [Mþ Hþ],
found 305.1284. tR = 6.27 min, purity = 98.2%.
Kinetics Assay for IC50 Determination. IC50 determinations
for wild type and mutants of EGFR were measured with the
HTRF KinEASE-TK assay from Cisbio according to the
manufacturer’s instructions. A biotinylated poly Glu-Tyr sub-
strate peptide was phosphorylated by EGFR. After completion
of the reaction, an antiphosphotyrosine 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
N-(3-Ethynylphenyl)-6,7-dimethoxyquinazolin-4-amine (4c).
Compound 4c was synthesized by following the procedure
described for 4a using, 4,4-chloro-6,7-dimethoxyquinazoline
(0.2 g, 0.9 mmol), 3-ethynylaniline (0.1 g, 0.9 mmol), and
catalytic amount of 2N HCl (50 μL). The crude was purified
over silica gel using CH2Cl2/MeOH (100:1 to 100:5) as an
eluent affording 0.25 g (92%) of 4c; mp 290-292 °C. 1H
NMR (400 MHz, MeOD) δ 8.73 (s, 1H), 7.99 (s, 1H), 7.88

2900 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Pawar et al.
€
substrate peptide. ATP concentrations were set at their respec-
tive Km values (30 μM for the EGFR-WT, 60 μM for EGFR-
Leu858Arg, and 30 μM for EGFR-Leu858Arg, Thr790Met),
and 50 nM of substrate were used for both wild type and mutant
EGFR. Kinase, substrate peptide, and inhibitor were preincu-
bated for 2 h before the reaction was started by addition of ATP.
A Tecan Safire2 plate reader was used to measure the fluores-
cence 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 (including no inhibitor) were
plotted against inhibitor concentrations and fit to a Hill 4-
parameter equation to determine IC50 values. Each reaction
was performed in duplicate, and at least three independent
determinations of each IC50 were made.
Structural Modeling. The small molecule structures of 4-
anilinoquinolines were sketched and minimized in DS ViewerPro
(http://www.accelrys.com) and aligned to the kinase domains of
the EGFR-erlotinib (PDB code: 1M17) and EGFR-3b com-
plexes (PDB code: 2J5F) using PyMol (http://www.pymol.org).
In the modeled 4-anilinoquinoline EGFR complexes, the in-
hibitors adopt binding modes isostructural to erlotinib or 3b in
EGFR. N1 of the quinoline scaffold forms one key hydrogen
bond to the backbone of the hinge region (Met793 in EGFR).
Cell Lines. NSCLC cells were obtained from ATCC (www.
atcc.org) and were maintained as described previously.23 Ba/F3
cell lines were established as described previously24 and were
maintained in RPMI media containing 10% FCS and 1% of
penicillin and streptomycin.
Viability Assays. The assay was set out as described pre-
viously.25 In brief, cells were plated into sterile 96-well plates
using a Multidrop instrument (www.thermo.com) and after
24 h of incubation a dilution series of compounds was added.
Viability was determined after 96 h using the Cell Titer-Glo
assay (www.promega.com). Luminescence was measured on a
Mithras LB940 plate reader (www.bertholdtech.com). Half-
maximal inhibitory concentrations were determined using the
R package “ic50”.
Immunoblotting. Immunoblotting was performed using stan-
dard procedures as described before.25 Briefly, cells were treated
for 24 h and concentration of a given compound prior to using whole
cell lysates for immunoblotting. The following antibodies were
utilized: p-EGFR (Biosource, USA), antirabbit-antibody, antimouse
(Millipore, Germany). The enhanced chemiluminescence (ECL)
system (Amersham-Pharmacia) was used to develop the blots.
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Acknowledgment. We thank Rogier Buijsman and Michael
Beck for helpful discussions. The work was supported by
the German Federal Ministry for Education and Research
through the German National Genome Research Network-
Plus (NGFN-Plus) (BMBF grant 01GS08102), all to D.R., and
by the Deutsche Krebshilfe (grant 107954), by the Fritz-
Thyssen-Stiftung (grant 10.08.2.175) as well as by the German
National Genome Research Network-Plus (NGFN-Plus)
(BMBF grant 01GS08100), all to R.K.T. W.A.L.vO. thanks
the Alexander von Humboldt Foundation for a Georg For-
ster ResearchFellowship for Experienced Researchers andthe
UniversityoftheWitwatersrandfor sabbaticalleave. Schering
Plough, Bayer-Schering Pharma, Merck-Serono, and Bayer
CropScience are thanked for financial support.
€
€
Supporting Information Available: Figure and experimental
data for compounds 7, 8a, 11a, 15a. This material is available
free of charge via the Internet at http://pubs.acs.org.
€
€
(16) Getlik, M.; Grutter, C.; Simard, J. R.; Kluter, S.; Rabiller, M.;
Rode, H. B.; Robubi, A.; Rauh, D. Hybrid compound design to
overcome the gatekeeper T338M mutation in cSrc. J. Med. Chem.
2009, 52, 3915–3926.
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