Discovery of boron-conjugated 4-anilinoquinazoline as a prolonged inhibitor of EGFR tyrosine kinase

Article abstract of DOI:10.1039/b909504g

Boron-conjugated 4-anilinoquinazolines were designed and synthesized as inhibitors of EGFR tyrosine kinase with possible covalent bond interactions between the boron atom and the nucleophilic groups of the EGFR kinase domain. Among the compounds synthesized, compounds 6c, 7b, and 7d reduced the EGF-mediated phosphorylation of EGFR tyrosine kinase and its downstream kinases including ERK and Akt in A431 cells. The cell growth was inhibited by these compounds through arrest of G1 cell cycle, which induced apoptosis. A time-dependent in vitro preincubation assay demonstrated the irreversible inhibition of compound 7d against EGFR tyrosine kinase. Quantum mechanical docking simulation revealed that the boronic acid moiety of compound 7d formed a covalent B-O bond with Asp800 in addition to hydrogen bonds with Asp800 and Cys797, which may cause the prolonged inhibition of compound 7d toward EGFR tyrosine kinase.

Full text of DOI:10.1039/b909504g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Discovery of boron-conjugated 4-anilinoquinazoline as a prolonged inhibitor
of EGFR tyrosine kinase†
Hyun Seung Ban,a Taikou Usui,a Wataru Nabeyama,a Hidetoshi Morita,b Kaori Fukuzawac and
Hiroyuki Nakamura*a
Received 14th May 2009, Accepted 15th July 2009
First published as an Advance Article on the web 20th August 2009
DOI: 10.1039/b909504g
Boron-conjugated 4-anilinoquinazolines were designed and synthesized as inhibitors of EGFR tyrosine
kinase with possible covalent bond interactions between the boron atom and the nucleophilic groups of
the EGFR kinase domain. Among the compounds synthesized, compounds 6c, 7b, and 7d reduced the
EGF-mediated phosphorylation of EGFR tyrosine kinase and its downstream kinases including ERK
and Akt in A431 cells. The cell growth was inhibited by these compounds through arrest of G1 cell
cycle, which induced apoptosis. A time-dependent in vitro preincubation assay demonstrated the
irreversible inhibition of compound 7d against EGFR tyrosine kinase. Quantum mechanical docking
simulation revealed that the boronic acid moiety of compound 7d formed a covalent B–O bond with
Asp800 in addition to hydrogen bonds with Asp800 and Cys797, which may cause the prolonged
inhibition of compound 7d toward EGFR tyrosine kinase.
However, despite the benefits of Iressa and Tarceva for treatment
Introduction
of NSCLC, most recurrent patients ultimately develop acquired
The signal transduction pathway through growth factor receptor
resistance to these agents. Recently, a mutation, T790M, within
tyrosine kinases is important for cell proliferation. Deregulation
of signaling pathway has been observed in many human tumors,
the EGFR kinase domain has been found in ~50% of these
patients, and therefore much attention has been paid for potency of
and thus these kinases have been investigated as potential targets
irreversible EGFR inhibitors, which have proven to be effective for
for cancer therapy.1 Epidermal growth factor receptor (EGFR)
the EGFR tyrosine kinase with T790M mutation.11,12 Among the
(also known as erb-B1 or Her-1) is a member of ErbB family
various 4-anilinoquinazoline derivatives synthesized,13–15 EKB-
of receptor tyrosine kinases, and the uncontrolled activation of
569 (phase II),16,17 HKI-272 (phase I),18 and CI-1033 (phase I)19,20
EGFR-mediated signaling may be due to overexpression of the
have been investigated as irreversible inhibitors. These compounds
receptors in numerous tumors including head and neck, lung,
have a Michael acceptor function, as a common structure,
breast, bladder, prostate, and kidney cancers, mutations resulting
substituted at the 6-position of the quinazoline ring to trap the
in constitutive activation in brain tumors, or overexpression of the
Cys-797 (Cys773 in an alternative numbering scheme) of EGFR,
ligands including EGF or TGF-a in pancreatic, prostate, lung,
which may cause the irreversible inhibition. According to X-ray
structural analysis of a complex with Tarceva, the thiol of Cys-797
ovary, and colon cancers.2,3
In 1994, Fry and co-workers discovered 4-anilinoquinazoline
in fact is located near the methoxyethoxy group substituted at the
(PD 153035) as a specific inhibitor toward EGFR tyrosine kinase.4
6-position of Tarceva,21,22 as shown in Fig. 1.
Since their discovery, various 4-anilinoquinazoline derivatives
We focused on the substituent at the 6-position and designed
have been synthesized, and ZD-1839 (IressaTM),5,6 and OSI-774
)
(TarcevaTM 7,8 have been developed as reversible binding inhibitors
boron-conjugated 4-anilinoquinazolines. A boron atom has a
vacant orbital and interconverts with ease between the neutral
sp2 and the anionic sp3 hybridization states, which generates
a new stable interaction between a boron atom and a donor
molecule through a covalent bond. Recently the X-ray crystal
structure of the 20S proteasome in complex with bortezomib
was reported.23 In this structure, boronic acid of bortezomib
covalently interacts with the Thr-1 hydroxyl in the active site of the
20S proteasome, forming the hybridized borate. Bond energies of
C–S, B–S, and B–O s-bonds have been estimated as 65, 24, and
18 kcal/mol, respectively,24,25 and that of hydrogen bonding is
3–10 kcal/mol.24,26 Therefore, if we can introduce a boron atom
into the 6-position of the quinazoline framework via a linker
of suitable length, as illustrated in Fig. 1A, a covalent bond
interaction would be expected between the boron atom and the
thiol of Cys797 or the carboxyl group of Asp800. This interaction,
which is stronger than the hydrogen bond but not as strong as the
of EGFR kinase activity and recently approved for non-small-cell
lung cancer (NSCLC) therapy. Furthermore, Lapatinib has been
investigated as a dual reversible ErbB inhibitor with potent activity
against EGFR and Her-2 kinases, and approved for breast cancer
therapy.9,10
aDepartment of Chemistry, Faculty of Science, Gakushuin University,
Tokyo, 171-8588, Japan. E-mail: hiroyuki.nakamura@gakushuin.ac.jp;
Fax: (+81)3-5992-1029; Tel: (+81)3-3986-0221
bGraduate School of Science and Engineering, Yamagata University,
Yamagata, 990-8560, Japan
cMizuho Information & Research Institute, Inc., Tokyo, 101-8443, Japan
† Electronic supplementary information (ESI) available: QM/MM-
optimized geometries for the docking mode of EGFR-compound 7d,
effects of ATP concentration on in vitro preincubation time-dependent
EGFR inhibition by compound 7d, and 1H NMR spectra for compounds
2–4. See DOI: 10.1039/b909504g
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Fig. 1 (A) Expected binding conformation of boron-conjugated quinazolines (black) based on the X-ray crystal structure of the EGFR kinase domain
in complex with Tarceva (gray, PDB code: 1M17). (B) Structures of reversible and irreversible inhibitors of EGFR tyrosine kinase.
C–S or C–O covalent bond,27 may affect the prolonged inhibition
of EGF-stimulated signal transduction.
the EGFR and inhibitor by hybrid QM/MM calculations in the
ONIOM scheme,29,30 employing a Hartree–Fock (HF) method for
the QM part and a UFF force field for the MM part. Binding
energies between the EGFR and inhibitors were also evaluated
by full QM treatment using ab initio fragment molecular orbital
(FMO) methods31–34 at the HF level. This FMO approach has
been successfully used to evaluate molecular interactions of bio-
macromolecules such as a binding affinity of ligands to the human
estrogen receptor.35,36
In this paper, we have introduced a boron moiety into the
anilinoquinazoline framework via various lengths of linkers,
˚
˚
such as methylene (1.68 A), propenyl (3.90 A), and benzyl
˚
(5.81 A) groups (Fig. 2). We synthesized various boron-conjugated
4-anilinoquinazolines and investigated their selective inhibition
of EGFR kinase, inhibition of the EGFR-stimulated signaling
pathway, and effects on cancer cell growth and cell cycle, as well
as their irreversible interaction properties.
Furthermore, in order to analyze the EGFR-inhibitor binding
forms and their interactions in detail, molecular simulations
were carried out based on quantum mechanical (QM) methods.
Classical molecular mechanical (MM) approaches based on an
empirical force field have been widely used for molecular simula-
tions of protein properties such as enzyme–inhibitor interactions.
However, such an approach is not sufficiently accurate in the
description of the nature of the interactions between molecules.
Also, the force field of sp3-hybridized boron atom has still not
been established. On the other hand, a QM approach based on ab
initio molecular orbital (MO) calculations has been successfully
used to understand the structure and chemical properties of
molecules including coordinate linkages of sp3-hybridized boron
atoms, and therefore the QM approach has potential for accurate
representation of molecular interactions that could aid drug
design.28 Here, we have investigated docking geometries between
Results and discussion
Chemistry
The synthesis of the boron-conjugated anilinoquinazolines
6a–d and 7b–d is shown in Fig. 2. Briefly, 5-hydroxyanthranilic
acid 1 was converted to the quinazolinone 2 in 70% yield in
three steps according to the reported procedure.37 Protection
of the hydroxy group of 2 with acetic anhydride gave the
6-acetoxyquinazoline 3, which underwent chlorination with POCl3
to give the 4-chloroquinazoline 4 in 66% yield. Displacement of 4
with 3-chloroaniline followed by deprotection of the acetyl group
afforded the 6-hydroxy-4-anilinoquinazoline 5, quantitatively.38
=
Various boronic ester groups, such as (RO)2BCH CHCH2Cl
(8 and 9), (RO)2BCH2Br (10),39 and (RO)2BC6H4CH2Br (13),
were synthesized and introduced into the 6-position of the
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Fig. 2 Synthesis of boron-conjugated 4-anilinoquinazolines.
Table 1 Effects of the boron-conjugated 4-anilinoquinazolines on tyro-
anilinoquinazoline 5 through ether bond formation to give the
corresponding anilinoquinazolines 6a–d in 7–51% yields. Depro-
tection of boronic esters 6a–d was carried out by using KHF2
in aqueous methanol to give the boronic acids 7b–d in 18–28%
yields.40
sine kinase activity of EGFR, HER2, Flt-1, and KDRa
Inhibition at 1 mM (%)/IC50 (mM)a
Compound
EGFR
HER2
Flt-1
KDR
6a
55/0.64 0.02
53/0.58 0.01
65/0.20 0.02
20/nd
63/0.27 0.02
45/nd
—/nd
—/nd
—/nd
—/nd
—/nd
—/nd
—/nd
—/nd
—/nd
—/nd
—/nd
—/nd
—/nd
—/nd
—/nd
—/nd
11/nd
6/nd
6b
Enzyme and cellular inhibitory activities
6c
—/nd
—/nd
—/nd
—/nd
—/nd
40/nd
6d
Inhibitory activity of the boron-conjugated 4-anilinoquinazolines
against EGFR, HER-2, Flt-1, and KDR tyrosine kinases, was
determined by measuring the levels of phosphorylation of the
tyrosine kinase-specific peptides in vitro.41 As shown in Table 1,
most of the boron-containing 4-anilinoquinazolines selectively
suppressed EGFR tyrosine kinase activity without inhibiting
HER-2, Flt-1 or KDR kinases, and their IC50 values against
EGFR tyrosine kinase were lower than 1 mM. These results
indicate that the conjugation of a boron moiety to the 6-position
of 4-anilinoquinazolines has a selective inhibitory activity toward
EGFR tyrosine kinase. Under our experimental conditions,
Tarceva, which was used as a positive control for enzyme assay, also
showed significant inhibition of EGFR tyrosine kinase (Table 1).
Among the boron-conjugated 4-anilinoquinazolines synthesized,
compounds 6d and 7c showed weak inhibitions of EGFR tyrosine
kinase (20 and 45%, respectively) at 1 mM. Boronic acids 7b
and 7d showed more potent inhibition compared to their ester
derivatives 6a, 6a, and 6d. Since the boron moiety may be located
in the hydrophilic region surrounded by Cys797 and Asp800 in
the binding formation to EGFR kinase, the boronic acids are
7b
7c
7d
55/0.85 0.03
82/0.06 0.07
Tarceva
a The drug concentrations are those required to inhibit the phosphorylation
of the poly(Glu:Tyr) substrate by 50% (IC50). They were determined from
semilogarithmic dose–response plots, and represent the mean s.d. of
triplicate samples. —, no inhibitory effect at 1 mM; nd, not determined.
preferable to the corresponding boron esters due to their steric
and hydrophilic properties. In contrast, boronic acid7c showed less
potent inhibition because the linker between the boronic acid and
the quinazoline ring may be too short to occupy the hydrophilic
region.
We next examined the effects of the boron-conjugated
4-anilinoquinazolines on the EGF-induced tyrosine phosphory-
lation of EGFR and the signaling cascades in A431 cells by
immunoblot analysis. Treatment of A431 with EGF (10 ng/ml)
rapidly induced autophosphorylation of EGFR, and the level of
the phosphorylation reached a maximum at 10 min after EGF
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stimulation (data not shown). Under these conditions, the boron-
conjugated 4-anilinoquinazolines potently suppressed the EGF-
induced phosphorylation of EGFR at 1 mM concentration of
compounds (Fig. 3A).
with compounds at various concentrations for 72 h and their cell
growth inhibition was determined by MTT assay. As shown in
Fig. 3C, the boron compounds reduced the proliferation of A431
cells, which overexpress EGFR, and their GI50 values were in the
range 0.54–2.59 mM. Lower cell growth inhibitions were observed
in other cell lines, which express a low level of EGFR. These
results indicate that the selective inhibition of A431 cell growth by
the boron-conjugated 4-anilinoquinazolines is a consequence of
the inhibition of EGFR tyrosine kinase.
Cell cycle G1 arrest and induction of apoptosis
To characterize the cell growth inhibition induced by the boron-
conjugated 4-anilinoquinazolines, effects of compounds on cell
cycle were investigated by flow cytometry analysis. Treatment
of A431 cells with compounds 6c, 7b, and 7d at 1 mM for
24 h led to profound changes in cell-cycle profiles as shown in
Fig. 4A. The boron-conjugated 4-anilinoquinazolines induced a
massive accumulation of cells in the G1 phase and decreased the
proportion of cells in G2/M phase. Tarceva also induced G1-arrest
with a similar potency. To further characterize the cell-death mech-
anism induced by the boron-conjugated 4-anilinoquinazolines,
a biparametric cytofluorimetric analysis was performed using
FITC-labeled annexin V and propidium iodide (PI), which stain
phosphatidylserine residue and DNA, respectively.43 A431 cells
Fig. 3 Inhibition of the EGF-induced phosphorylation of EGFR and
its downstream kinases, and cell growth. (A) and (B) A431 cells were
incubated with the boron-conjugated 4-anilinoquinazolines (1 mM) or
Tarceva (0.3 mM) and then stimulated with EGF (10 ng/mL). The levels
of each kinase were detected by immunoblot analysis with the specific
antibody. (C) A431, HepG2, KatoIII, or SKBR3 cells were incubated
for 72 h with various concentrations (10 nM to 10 mM) of compounds
and then the rates of viable cells were determined by MTT assay. The drug
concentration required to inhibit cell growth by 50% (GI50) was determined
from semilogarithmic dose–response plots, and results represent the mean
s.d. of triplicate samples.
It has been reported that the autophosphorylation of EGFR
tyrosine kinase activated various downstream kinases including
ERK and Akt, which play an important role in the regulation
of cell proliferation and apoptosis, respectively.42 Therefore, we
examined the effects on the EGFR-dependent activation of
downstream signaling pathways. As shown in Fig. 3B, the boron-
conjugated 4-anilinoquinazolines, except compounds 6d and 7c,
also significantly suppressed the EGF-induced phosphorylation
of ERK and Akt in parallel with the inhibition of EGFR
autophosphorylation. These results indicate that the boron-
conjugated 4-anilinoquinazolines also induce the inhibitory effect
on autophosphorylation of EGFR tyrosine kinase in cells as well
as EGFR kinase in vitro, and arrest the downstream signaling
pathway.
To demonstrate whether the inhibitory activity of the boron-
conjugated 4-anilinoquinazolines toward EGFR tyrosine kinase
is implicated in cell growth, we examined the effects of the
compounds on proliferation of various human carcinoma cell lines
(A431, HepG2, KatoIII, and SKBR3). The cells were incubated
Fig. 4 Induction of cell cycle G1 arrest and apoptosis. A431 cells
were incubated for 24 h with the boron-conjugated 4-anilinoquinazolines
(1 mM) or Tarceva (0.3 mM). (A) The cells were harvested and stained with
propidium iodide (PI). (B) The cells were stained with annexin V-FITC and
PI. The cell cycles and apoptosis were determined by a flow cytometer.
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were treated with the compounds for 24 h and then stained
with PI and FITC-labeled annexin V. The resulting green (FITC)
and red (PI) fluorescence was monitored by flow cytometry.
As indicated in Fig. 4B, the number of annexin V-positive and
PI-negative cells increased by the treatment with compounds 6c,
7b, 7d, and Tarceva (the area labelled D). These results indicate
that the boron-conjugated 4-anilinoquinazolines possess not only
antiproliferative but also apoptotic activities in the A431 cell
growth inhibition through the suppression of EGFR tyrosine
kinase activity.
dependent inhibition of compound 7d against the EGF-induced
phosphorylation of EGFR was observed in A431 cells (Fig. 5B).
These results also support the irreversible inhibitory property
of compound 7d. We next investigated the prolonged inhibition
implicated in continuous reduction of cell growth. As shown in
Fig. 5C, MTT assay revealed that compound 7d showed retentive
reduction of cell growth after removal of compounds from the cell-
medium by washing even though its inhibitory activity was not
potentially high in comparison with other compounds, whereas
the inhibitory effects of other compounds including Tarceva were
essentially lost upon washing. These results indicate that continued
reduction of cell growth by compound 7d may be due to the
irreversible inhibition of EGFR tyrosine kinase. Fig. 5D shows
dose-dependent growth inhibition curves of cells treated with
compound 7d with and without washing, and similar values for
GI50 were observed in both cases. It was reported that the lack of
autophosphorylation sites resulted in reduction of internalization
and degradation rates of EGFR.44 Therefore, the irreversible in-
hibition of EGFR autophosphorylation by compound 7d induces
prolonged reduction effects of internalization and degradation
rates of EGFR, which may lead to the retentive reduction of cell
growth.
Prolonged inhibition by irreversible binding to EGFR tyrosine
kinase in cells
The prolonged inhibition ability of the boron-conjugated
4-anilinoquinazolines toward EGFR tyrosine kinase was inves-
tigated by experiment using A431 exposed to the compounds and
washed prior to the assay.15 After continuous incubation of A431
cells with the boron-conjugated 4-anilinoquinazolines at 1 mM
or Tarceva at 0.3 mM for 1 h followed by EGF stimulation, each
compound inhibited autophosphorylation of EGFR in A431 cells,
as shown by immunoblot analysis (Fig. 5A). When the cells were
washed to remove unbound compounds from the cell-medium
and incubated for an additional 5 h prior to EGF stimulation,
the activity of EGFR autophosphorylation was restored, while
for cells treated with compound 7d, the autophosphorylation
was still inhibited. These results suggest that compound 7d
irreversibly inhibited to EGFR tyrosine kinase, whereas other
compounds, such as 6c, 7b, and Tarceva, reversibly interacted
to EGFR tyrosine kinase. Furthermore, the preincubation time-
In vitro irreversible inhibition of EGFR tyrosine kinase activity
To clarify the irreversible inhibition of EGFR tyrosine kinase
by the boron-conjugated 4-anilinoquinazoline in detail, in vitro
time-dependent inhibition assay was performed.45 As shown in
Fig. 6, EGFR irreversible inhibitor PD168393 at 10 nM inhibited
EGFR tyrosine kinase activity in a time-dependent manner;
however, the inhibitory activity of reversible inhibitor Tarceva at
10 nM plateaued after incubation for 5 to 15 min. Under these
conditions, compound 7d inhibited EGFR tyrosine kinase activity
in a concentration- and time-dependent manner (Fig. 6). These
results suggest that the boron-conjugated 4-anilinoquinazoline 7d
irreversibly suppresses EGFR tyrosine kinase.
Fig. 5 Inhibition of the EGF-induced phosphorylation of EGFR and
cell growth. (A) A431 cells were incubated with the boron-conjugated
4-anilinoquinazolines (1 mM) or Tarceva (Tar, 0.3 mM), and then stimulated
with EGF (10 ng/mL) or washed with fresh medium for 5 h prior to
stimulation of EGF. The levels of each kinase were detected by immunoblot
analysis with the specific antibody. (B) Time-dependent inhibition of
EGF-induced phosphorylation of EGFR by compound 7d. A431 cells were
incubated with the indicated time and concentration of compound 7d, and
then stimulated with EGF (10 ng/mL). (C) A431 cells were incubated for
18 h with compounds 6c (1 mM), 7b (1 mM), 7d (3 mM) or Tarceva (0.3 mM)
and then washed with fresh medium. After further incubation for 54 h, the
rates of viable cells were determined by MTT assay. (D) Dose-dependent
cell growth with or without wash of cells treated with compound 7d was
determined.
Fig. 6 Preincubation time-dependent inhibition of EGFR by compound
7d. EGFR was incubated in kinase assay buffer with various concentra-
tions of compound 7d, 10 nM Tarceva (Tar), or 10 nM PD168393 (PD).
After incubation for the indicated time, kinase assay was initiated by adding
ATP (10 mM), and phosphorylation of the poly(Glu:Tyr) substrate was
detected. The drug concentrations required to inhibit the phosphorylation
of the poly(Glu:Tyr) substrate by 50% (IC50) were determined from
semilogarithmic dose–response plots, and results represent the mean s.d.
of triplicate samples.
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Recently, the X-ray cocrystal structure of EGFR and PD168393
was reported and a covalent bond has been observed be-
tween the acrylamide Michael acceptor on PD168393 and
the targeted Cys797 in the complex (PDB code: 2JF5).21
We tried to detect the Cys797-containing peptide fragment
VQLITQLMPFGCLLDYVREHKD, which was generated by
Molecular docking and quantum mechanical simulation
We performed a molecular docking simulation of compound 7d
with the ATP-binding site of EGFR kinase domain (PDB code
1M17). The initial structure of protein backbone was taken from
the X-ray structure of EGFR kinase domain in complex with
Tarceva.22 A conformation search for three rotatable bonds of
compound 7d (Fig. 7A) was performed to examine the boron
docking mode toward surrounding residues. As shown in Fig. 7B,
65 conformers were observed and classified into three clusters.
The boronic acid of compound 7d was located around Cys797
or Asp800 in the clusters 1 and 2, respectively, suggesting
interactions of the boronic acid with such residues. Based on
these two conformations, three docking models (models 1–3) were
constructed for further calculations. The initial structure of model
1 was selected from the cluster 2, and those of model 2 and model
3 were selected from the cluster 1. The thiol group of Cys797 was
in neutral form (–SH) for model 1 and model 2, and negatively
charged form (–S-) for model 3.
The docking structure of Tarceva (X-ray structure) and the
three models of compound 7d were further optimized by using
the ONIOM-QM/MM simulation at the HF/6-31G(d):UFF
level of theory. As shown in Fig. 8, the QM region includes
both interaction sites of quinazoline ring and boronic acid moi-
eties: binding site residues of EGFR, a hydrogen-bonding water
molecule, and inhibitor. Fig. 9 shows the three docking modes
¯
treated with trypsin digestion of EGFR kinase domain, by
ESI-TOF and MALDI-TOF mass spectroscopies in order to
clarify the covalent bond interaction of compound 7d and
EGFR. However, this peptide fragment was not ionized enough
to be detected by the mass spectroscopy under our tested
conditions. In fact, no report has been found for detection of
this peptide fragment by mass spectroscopic analysis. Fry and
coworkers reported that the covalent conjugation of Cys773 in
the fragment EILDEAYVMASVDNPHVCR with PD168393 was
¯
detected by ESI mass spectroscopy.14 However, Cys773 is located
near the aniline ring of both inhibitors Tarceva and PD168393
in the reported cocrystal structures.21,22 Therefore, the report for
detection of thecovalentconjugation ofCys773and PD168393 has
exhibited the possibility of alternative binding mode of PD168393
in EGFR kinase domain. Since mass-spectroscopic analysis
was not been suitable for clarification of the covalent bonding
interaction of Cys797 and compound 7d, we next demonstrated
molecular docking and quantum mechanical simulation to predict
the plausible binding mode of compound 7d in the EGFR kinase
domain.
Fig. 7 Conformation search results of compound 7d. (A) Rotatable bonds used for conformation search of compound 7d. (B) The line representation
refers to searched inhibitor structure: the stick representation refers to Cys797, Asp800, and initial geometry of inhibitor: the trace line representation is
the Ca backbone.
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Table 2 Calculated binding energies (in kcal/mol) between EGFR and
inhibitors
Compound
Boron hybridization
DE
7d – model 1
7d – model 2
7d – model 3
Tarceva
sp3
sp2
sp2
—
-90.2190
-50.9435
-56.3295
-33.6273
sp2 for models 2 and 3. A strong hydrogen bond between hydroxyl
hydrogen of boronic acid and carboxyl oxygen atom of Asp800
˚
was observed, with lengths of 1.60 and 1.73 A for models 2 and 3,
respectively. A second hydrogen bond was also observed in model
Fig. 8 High-level quantum mechanical (QM) region in the ONIOM
calculation. Both side chains and backbones were capped at the Ca
positions (dotted curves) by using hydrogen link atoms. Dotted lines
indicate hydrogen bonds between the EGFR and inhibitor.
3 between the other hydroxyl hydrogen of boronic acid and thiol
-
˚
ion S of Cys797, with a length of 2.43 A. Therefore, the interaction
between the EGFR and inhibitor at the boronic acid site is even
stronger than that of the quinazoline ring site, and this covalent
interaction may induce the prolonged inhibition of compound 7d
toward EGFR tyrosine kinase.
For a quantitative evaluation of the EGFR–inhibitor interac-
tion, binding energies of each binding mode were calculated based
on the ab initio FMO method. The energy of each of the three
systems, i.e., the receptor (EEGFR), the inhibitor (Elinhibitor), and their
complex (Ecomplex) were calculated, where the hydrogen-bonded
water molecule was included in the receptor. The binding energy,
DE, can be obtained by the following equation:
DE = Ecomplex - (EEGFR + Elinhibitor
)
(1)
The calculated DE values are summarized in Table 2. For all
binding models of compound 7d, absolute values of DE were larger
than that of Tarceva due to additional covalent and hydrogen
bonds at the boronic acid site. The strongest interaction between
the EGFR and 7d was observed for model 1 with a DE value of
-90.2 kcal/mol. The existence of a B–O covalent bond between
boronic acid and Asp800 supports such a strong interaction.
Model 2 and model 3 indicate a medium degree of interactions,
-50.9 and -56.3 kcal/mol, respectively, hydrogen bonds between
the boronic acid and Cys797 and Aps800 causing the stabilization.
Therefore, model 1 is energetically preferred as the binding form
of compound 7d toward EGFR tyrosine kinase.
Fig. 9 Binding modes of compound 7d (models 1–3) overlaid with the
binding conformation of Tarceva into the active site of EGFR kinase
domain. Model 1, model 2 and model 3 are shown with backbone colors
of pink, blue, and yellow, respectively.
of the compound 7d (models 1–3) into the active site of EGFR
kinase domain overlaid with the binding conformation of Tarceva
(schematic figures and geometrical values for each docking mode
are available in the ESI†). According to the simulation, the
4-anilinoquinazoline moieties of both compound 7d and Tarceva
bind to the kinase domain in a similar manner. In all binding
structures, three hydrogen bonds were observed as well as the
X-ray structure: one direct hydrogen bond between the nitrogen
(N1) of the quinazoline ring and the amide nitrogen of Met793
Conclusions
˚
(N–H lengths 2.28–2.44 A); two hydrogen bonds mediated by
In the present study, we designed boron-conjugated 4-anilino-
quinazolines as inhibitors of EGFR tyrosine kinase with possible
covalent bond interactions between the boron atom and the
nucleophilic thiol of the EGFR kinase domain. Selective inhibition
of boron-conjugated 4-anilinoquinazolines was observed at 1 mM;
compound 6c exhibited significant inhibition of EGFR tyrosine
kinase with an IC50 value of 0.19 mM. Boron compounds also
reduced the proliferation of A431 cells, selectively, with GI50
values of 0.53–2.42 mM. Among the compounds synthesized,
compounds 6c, 7b, and 7d significantly suppressed the EGF-
induced phosphorylation of ERK and Akt in parallel with the
inhibition of EGFR autophosphorylation in A431 cells, and
compound 7d was found to possess prolonged inhibitory activity
toward autophosphorylation of EGFR tyrosine kinase at 5 h
after cell wash, although inhibition of the cell growth was not
single water molecule among the nitrogen (N3) of the quinazoline
ring, the thiol hydrogen of Cys775, and hydroxyl oxygen of Thr790.
The corresponding calculated values are 2.28–2.40 A between N3
and the water hydrogen; 2.32–2.65 A between the thiol hydrogen
˚
˚
˚
of Cys775 and the water oxygen; 2.02–2.05 A between the hydroxyl
oxygen of Thr790 and the water hydrogen. Also, the chloro group
on the aniline ring of compound 7d is located in a very similar
position to the ethynyl group of Tarceva. In the binding structures
of compound 7d (models 1–3), additional bonds were observed
at the boronic acid site. Model 1 indicated a covalent B–O bond
between the sp3-hybridized boron atom and the carboxyl oxygen
atom of Asp800. The calculated B–O distance is 1.58 A, which is
longer than other two B–O lengths in the boronic acid (1.44 and
1.42 A). In contrast, the hybridization state of boron atom was
˚
˚
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particularly high in comparison with other compounds. Flow
cytometry analysis of compounds 6c, 7b, and 7d revealed that
these compounds induced G1 arrest in cell cycle and apoptosis.
Docking studies suggest that the quinazoline ring of compound 7d
is bound to the EGFR kinase domain in a very similar manner to
that of Tarceva. The boronic acid moiety of compound 7d is shown
to be able to form a covalent B–O bond with Asp800 and hydrogen
bonds with Cys797 and Asp800, which may cause the prolonged
inhibition of EGFR tyrosine kinase by compound 7d. Although
the acidity of each boronic acid and physico-chemical nature of
aromatic and aliphatic boronic acids should affect the binding
to the protein, these results suggest that boron has potential in
pharmaceutical drug design for prolonged interaction to target
proteins through covalent bonds.
at 0 ◦C and the reaction was allowed to warm at room temperature
for 1 h. The solvent was removed under reduced pressure and the
residue was purified by silica gel column chromatography with
dichloromethane/methanol (20:1) to give 3 (1.26 g, 95%) as a
1
yellow solid: H NMR (400 MHz, CDCl3) d = 10.95 (bs, 1 H),
8.97 (s, 1 H), 8.01 (d, J = 2.8 Hz, 1 H), 7.80 (d, J = 8.8 Hz,
1 H), 7.54 (dd, J = 8.8 Hz, 2.8 Hz, 1 H), 2.36 (s, 3 H); 13C NMR
(75 MHz, CDCl3) d = 169.0, 149.4, 146.7, 142.9, 129.3, 129.1,
123.4, 118.4, 20.9; IR (KBr) 3067, 2910, 1684, 1653, 1434, 1229,
835 cm-1; MS(ESI) m/z 227 [M+Na]+.
4-Chloroquinazolin-6-yl acetate (4)
To a solution of 3 (1.15 g, 5.64 mmol) in dichloromethane (40 ml)
were added POCl3 (0.64 ml, 6.8 mmol) and ethyl diisopropylamine
(1.27 ml, 14 mmol) and the mixture was refluxed for 12 h. The
solvent was removed under reduced pressure and the residue was
purified by silica gel column chromatography with hexane/ethyl
acetate (1/1) to give 4 (1.0 g, 80%) as a yellow solid:1H NMR
(400 MHz, CDCl3) d = 9.05 (s, 1 H), 8.11 (d, J = 8.8 Hz, 1 H),
8.01 (d, J = 2.4 Hz, 1 H), 7.73 (dd, J = 8.8, 2.4 Hz, 1 H), 2.40 (s,
3 H); 13C NMR (75 MHz, CDCl3) d = 168.9, 162.0, 153.5, 150.1
149.1, 130.5, 124.5, 116.8, 21.0; IR (KBr) 1869, 1757, 1684, 1653,
1558, 1508, 1205, 1177 cm-1; MS(ESI) m/z 245 [M+Na]+.
Experimental
General methods
1H NMR and 13C NMR spectra were measured on JEOL
JNM-AL 300 (300 MHz) and VARIAN UNITY-INOVA 400
1
(400 MHz) spectrometers. Chemical shifts of H NMR and 13C
NMR are expressed in parts per million (ppm, d units), and
coupling constants are expressed in units of hertz (Hz). IR
spectra were measured on a Shimadzu FTIR-8200A spectrometer.
Analytical thin-layer chromatography (TLC) was performed on
glass plates (Merck Kieselgel 60 F254 or RP-18 F254, layer thickness
0.2 mm). Samples were visualized by UV light (254 nm), I2,
or KMnO4. Column chromatography was performed on silica
gel (Merck Kieselgel 70–230 mesh). All reactions were carried
out under argon atmosphere using standard Schlenk techniques.
Most chemicals were of analytical grade and used without further
purification.
4-(3-Chloroanilino)-6-hydroxyquinazoline (5)
To a solution of 4 (839 mg, 3.8 mmol) in isopropanol (5 ml)
was added 3-chloroaniline (1.0 ml, 9.4 mmol) and the mixture
was refluxed for 12 h. The solvent was removed under reduced
pressure and the residue was dissolved in methanol (20 ml). The
25% aqueous ammonia solution (4 ml) was added and the mixture
was stirred at room temperature for 12 h. The solvent was removed
until the solid was precipitated and the resulting solid was filtered,
washed with dichloromethane (20 ml), and dried in vacuo to give 5
(1.0 g, >99%) as a pale yellow solid:1H NMR (400 MHz, CD3OD)
d = 8.35 (s, 1 H), 7.88 (t, J = 1.6 Hz, 1 H), 7.59 (d, J = 9.2 Hz, 1
H), 7.55 (d, J = 2.4 Hz, 1 H), 7.33 (dd, J = 2.4 Hz, 9.2 Hz, 1 H),
7.26 (t, J = 8.0 Hz, 1 H), 7.05 (dt, J = 2.0, 8.0 Hz, 1 H);13C NMR
(75 MHz, CD3OD3) d = 159.2, 158.1, 152.9, 145.0, 136.3, 135.5,
131.2, 129.8, 126.0, 125.4, 123.8, 122.1, 118.2, 106.0; IR (KBr)
2654, 1622, 1595, 1570, 1541, 1447, 1396 cm-1; MS(ESI) m/z 272
[M + H]+; Analysis calculated for C17H10ClN3O: C, 61.89; H, 3.71;
N, 15.47. Found: C, 58.25; H, 3.55; N, 15.57.
6-Hydroxy-4(1H)-quinazolinone (2)
To a solution of hydroxyanthranilic acid 1 (12.7 g, 83 mmol) in
THF (300 ml) was added ethyl chloroformate (23.8 ml, 249 mmol)
and the reaction mixture was refluxed for 24 h. The solvent
was removed under reduced pressure. The resulting residue was
dissolved in diethyl ether (300 ml) and PBr3 (15.3 ml, 84 mmol)
was added. The mixture was stirred at 50 ◦C for 24 h and cooled
to room temperature. After addition of water (200 ml), the white
precipitate was filtered and dried in vacuo. The resulting solid
was dissolved in formamide (198 ml, 5.0 mol) and the reaction
mixture was refluxed for 24 h. After the mixture was cooled to
room temperature, water (100 ml) was added. The precipitate was
filtered and washed with water (100 ml) to give 2 (9.45 g, 70%) as a
yellow solid: 1H NMR (300 MHz, CD3OD) d = 7.87 (s, 1H), 7.48
(d, J = 6.0 Hz, 1H), 7.41 (d, J = 3.0 Hz, 1H), 7.21 (dd, J = 6.0,
3.0 Hz, 1H);13C NMR (75 MHz, CD3OD) d = 163.5, 158.7, 143.7,
134.7, 129.7, 125.8, 125.1, 110.4; IR (KBr) 3078, 1670, 1653, 1508,
1489, 1340, 1248 cm-1; HRMS(ESI) calcd for C8H7N2O2 [M +
H]+: m/z 163.0508, found m/z 163.0503.
Synthesis of boronic esters (8–13)
2-(3-Chloropropenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane
(8).
A
mixture of trans-3-chloro-1-propenylboronic acid
(120 mg, 1.0 mmol) and pinacol (118 mg, 1.0 mmol) in Et2O
(5 ml) was stirred at room temperature for 24 h. The mixture was
dried over MgSO4 and concentrated to give 8 (202 mg, 99%) as a
1
yellow oil: H NMR (300 MHz, CDCl3) d = 6.64 (dt, J = 18.0,
6.0 Hz, 1H), 5.74 (dt, J = 18.0, 3.0 Hz, 1H), 4.10 (dd, J = 6.0,
3.0 Hz, 2H), 1.25 (s, 12H); 13C NMR (75 MHz, CDCl3) d = 146.6,
83.5, 83.2, 46.0, 24.7; IR (neat) 3404, 2980, 2934, 1643, 1475, 1358,
1146,851 cm-1; MS(ESI) m/z 203 [M + H]+; Analysis calculated
for C9H16BClO2: C, 53.38; H, 7.96. Found: C, 53.34; H, 7.90.
3,4-Dihydro-4-oxoquinazolin-6-yl acetate (3)
To a solution of 2 (1.0 g, 6.5 mmol) and triethylamine (0.2 ml,
1.3 mmol) in dichloromethane (40 ml) were added acetic anhydride
(1.8 ml, 19 mmol) and dimethylaminopyridine (24 mg, 0.2 mmol)
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4-(3-Chloropropenyl)-2,9,9-trimethyl-3,5-dioxa-4-boratricyclo-
[6.1.1.02,6]decane (9). Synthesized from trans-3-chloro-1-
propenylboronic acid (602 mg, 5.0 mmol) and (1S, 2S, 3R,
5S)-(+)-pinandiol (851 mg, 5.0 mmol) using the procedure
described for 8 to give 9 (624 mg, 49%) as a yellow oil:1H NMR
(400 MHz, CDCl3) d = 6.64 (dt, J = 18.0, 6.0 Hz, 1 H), 5.77 (dt,
J = 18.0, 10.2 Hz, 1 H), 4.32 (dd, J = 8.8, 1.6 Hz, 1 H), 4.11 (dd,
J = 6.0, 1.2 Hz, 2 H), 2.35 (ddt, J = 14.4, 8.8, 2.0 Hz, 1 H), 2.22
(m, 1 H), 2.07 (t, J = 6.0 Hz, 1 H), 1.85–1.94 (m, 2 H), 1.41 (s,
3 H), 1.19 (d, J = 10.4 Hz, 1 H), 1.29 (s, 3 H), 0.85 (s, 3 H); 13C
NMR (75 MHz, CDCl3) d = 146.5, 101.3, 85.9, 77.9, 51.2, 46.0,
39.4, 38.1, 35.3, 28.5, 27.0, 26.4, 23.9; IR (neat) 2920, 2872, 1643,
1362, 1283, 1030, 851 cm-1; Analysis calculated for C13H20BClO2:
C, 61.34; H, 7.92. Found: C, 61.44; H, 7.88.
2.26 (m, 1 H), 2.15 (t, J = 6.0 Hz, 1 H), 1.84–2.06 (m, 2 H), 1,48
(s, 3 H), 1,31 (s, 3 H), 1,21 (d, J = 10.8 Hz, 1 H), 0.89 (s, 3 H);
13C NMR (75 MHz, CDCl3) d = 135.1, 126.1, 125.8, 101.3, 86.2,
78.2, 65.3, 51.4, 39.5, 38.1, 35.5, 28.6, 27.0, 26.4, 24.0; IR (neat)
3433, 2920, 2870, 1614, 1402, 1362, 1236, 1092, 1020, 820 cm-1;
Analysis calculated for C17H23BO3: C, 71.35; H, 8.10. Found: C,
71.41; H, 8.15.
4-(4-Bromomethylphenyl)-2,9,9-trimethyl-3,5-dioxa-4-boratri-
cyclo[6.1.1.02,6]decane (13). To a solution of 12 (1.0 g, 3.5 mmol)
in toluene (20 ml) was added phosphorus tribromide (0.34 ml,
3.5 mmol) at 0 ◦C and the reaction was allowed to warm at
room temperature. After 3 h, water (30 ml) was added and the
reaction mixture was then extracted with dichloromethane (2 ¥
50 ml). The combined organic layer was dried over MgSO4 and
concentrated. Purification by silica gel column chromatography
with hexane/ethyl acetate (1/1) gave 13 (658 mg, 53%) as a green
4-Bromomethyl-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo-
[6.1.1.02,6]decane ((RO)2BCH2Br, 10). To a solution of dibro-
moethane (4.2 ml, 6.0 mmol) and triisopropylborate (6.0 mmol)
in THF (30 ml) was added n-BuLi (1.6 M in hexanes, 1.4 ml,
6.0 mmol) at -80 ◦C with stirring. After 30 min, methanesulfonic
acid (0.4 ml, 6.0 mmol) was added at -80 ◦C and then the reaction
mixture was stirred at room temperature for 24 h. Solvent was
removed under reduced pressure and the residue was purified
by silica gel column chromatography with hexane/ethyl acetate
(10/1) to give 10 (1.5 g, 92%) as a colorless oil: 1H NMR (400 MHz,
CDCl3) d = 4.38 (dd, J = 8.8, 1.6 Hz, 1 H), 2.64 (s, 2 H), 2.36
(m, 1 H), 2.26 (m, 1 H), 2.08 (t, J = 8.4 Hz, 1 H), 1.87–1.98 (m, 2
H), 1.48 (m, 3 H), 1.31 (m, 3 H), 1.19 (d, J = 10.8 Hz, 1 H), 0.89
(s, 3 H); 13C NMR (75 MHz, CDCl3) d = 86.8, 78.6, 51.2, 39.3,
38.2, 35.2, 28.3, 26.9, 26.3, 23.9; IR (neat) 2922, 2872, 1416, 1384,
1339, 1286, 1030, 845 cm-1; Analysis calculated for C11H18BBrO2:
C, 48.40; H 6.65. Found: C, 48.28; H, 6.70.
1
oil: H NMR (300 MHz, CDCl3) d = 7.79 (d, J = 9.0 Hz, 2 H),
7.40 (d, J = 9.0 Hz, 2 H), 4.50 (s, 2 H), 4.45 (d, J = 9.0 Hz, 1 H),
2.42 (m, 1 H), 2.25 (m, 1 H), 2.15 (t, J = 6.0 Hz, 1 H), 1.90–2,04
(m, 2 H), 1.48 (s, 3 H), 1.31 (s, 3 H), 1.20 (d, J = 9.0 Hz, 1 H), 0.89
(s, 3 H); 13C NMR (75 MHz, CDCl3) d = 140.6, 135.3, 128.3, 86.3,
78.3, 51.3, 39.5, 38.1, 35.5, 33.3, 28.6, 27.0, 26.4, 24.0; IR (neat)
2920, 2870, 1614, 1402, 1362, 1236, 1091, 1022 cm-1; MS(ESI) m/z
373 [M+Na]+; Analysis calculated for C17H22BBrO2: C, 58.49; H
6.35. Found: C, 58.25; H, 6.62.
4-(3-Chloroanilino)-6-[3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-
2-yl)-allyloxy]quinazoline (6a)
To a solution of sodium hydride (60% in oil, 32 mg, 0.8 mmol)
in N,N¢-dimethylformamide (DMF, 2 ml) was added 5 (107 mg,
0.4 mmol) at 0 ◦C and the reaction was allowed to warm at room
temperature. After 30 min, the solution of boronic ester 8 (81 mg,
0.4 mmol) in DMF (3 ml) was added and the mixture was stirred
for 2 h. Water (10 ml) was added and the reaction mixture was
extracted with diethyl ether (50 ml). The solvent was removed
under reduced pressure and the residue was purified by silica gel
column chromatography with hexane/ethyl acetate (1/1) to yield
6a (12mg, 7%) as a yellow solid:1H NMR (300 MHz, CDCl3): d =
8.72 (s, 1 H), 7.92 (s, 1 H), 7.85, (d, J = 9.0 Hz, 1 H), 7.60 (d,
J = 9.0 Hz, 1 H), 7.84 (dd, J = 3.0, 9.0 Hz, 1 H), 7.60 (d, J =
9.0 Hz, 1 H), 7.48 (dd, J = 9.0, 3.0 Hz, 1 H), 7.33 (t, J = 9.0 Hz,
1 H), 7.14 (d, J = 9.0 Hz, 1 H), 7.10 (s, 1 H), 6.79 (dt, J = 18.0,
3.0 Hz, 1 H), 5.88 (d, J = 18.0 Hz, 1 H), 4.77 (d, J = 3.0 Hz, 2
H), 1.27 (s, 12 H); 13C NMR (75 MHz, CDCl3): d = 157.0, 156.4,
152.7, 145.9, 139.6, 134.7, 130.7, 130.0, 124.6, 124.3, 121.6, 119.5,
115.5, 101.0, 83.6, 77.2, 69.9, 24.8; IR (KBr) 2980, 2932, 1599,
1576, 1525, 1506, 1431, 1373, 1225, 1142, 839cm-1; MS(ESI) m/z
438 [M + H]+; Analysis calculated for C23H25BClN3O3: C, 63.11;
H, 5.76; N, 9.60. Found: C, 63.40; H, 5.96; N 9.32.
4-(2,9,9-Trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.02,6]dec-4-yl)-
benzaldehyde (11). Synthesized from 4-formylphenylboronic
acid (750 mg, 5.0 mmol) and (1S,2S,3R,5S)-(+)-pinandiol
(851 mg, 5.0 mmol) using the procedure described for 8 to give 11
1
(1.42 g, 99%) as a white solid: H NMR (400 MHz, CDCl3) d =
10.06 (s, 1 H), 7.98 (d, J = 9.0 Hz, 2 H), 7.87 (d, J = 9.0 Hz, 2 H),
4.48 (d, J = 9.0 Hz, 1 H), 2.43 (m, 1 H), 2.23 (m, 1 H), 2.17 (t, J =
6.0 Hz, 1 H), 1.95–2.02 (m, 2 H), 1.51 (s, 3 H), 1.32 (s, 3 H), 1.19
(d, J = 12.0 Hz, 1 H), 0.90 (s, 3 H); 13C NMR (75 MHz, CDCl3)
d = 192.6, 138.0, 135.2, 128.7, 86.7, 78.5, 51.3, 39.4, 38.2, 35.4,
28.6, 27.0, 26.4, 24.0; IR (KBr) 2976, 2916, 2731, 1705, 1508,
1350, 1205, 1088, 827 cm-1. Analysis calculated for C17H21BO3: C,
71.86; H, 7.45. Found: C, 72.15; H, 7.60.
[4-(2,9,9-Trimethyl-3,5-dioxa-4-boratricyclo[6.1.1.02,6]dec-4-yl)-
phenyl]methanol (12). To a solution of 11 (1.42 g, 5.0 mmol)
in methanol (30 ml) was added NaBH4 (473 mg, 12 mmol) at
-40 ◦C and the reaction mixture was stirred at room temperature
for 1 h. The solvent was removed under reduced pressure, and
the residue was dissolved in water (30 ml) and extracted with
dichloromethane (2 ¥ 50 ml). The combined organic layer was
dried over MgSO4 and concentrated. Purification by silica gel
column chromatography with hexane/ethyl acetate (1/1) gave 12
4-(3-Chloroanilino)-6-[3-(2,9,9-trimethyl-3,5-dioxa-4-
boratricyclo[6.1.1.02,6]dec-4-yl)-allyloxy]quinazoline (6b)
Synthesized from 5 (381 mg, 1.5 mmol), 9 (407 mg, 1.5 mmol)
using the procedure described for 6a, to give 6b (145 mg, 19%)
1
(1.0 g, 71%) as a colorless oil: H NMR (400 MHz, CDCl3) d =
1
7.81 (d, J = 8.0 Hz, 2 H), 7.38 (d, J = 8.0 Hz, 2 H), 4.72 (d,
J = 6.0 Hz, 2 H), 4.45 (dd, J =2.0, 8.8 Hz, 1 H), 2.41 (m, 1 H),
as a yellow solid: H NMR (400 MHz, CDCl3) d = 8.71 (s, 1H),
7.78–7.87 (m, 3 H), 7.58 (d, J = 8.0 Hz, 1 H), 7.46 (dd, J = 9.2,
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2.4 Hz, 1 H), 7.29 (t, J = 8.0 Hz, 1 H), 7.21 (d, J = 2.4 Hz, 1 H),
7.10 (dt, J = 8.0, 0.8 Hz, 1 H), 6.74 (dt, J = 18.0, 4.4 Hz, 1 H),
5.84 (d, J =18.0 Hz, 1 H), 4.65 (d, J = 4.4 Hz, 2 H), 4.32 (d, J =
8.4 Hz, 1 H), 2.35 (m, 1 H), 2.20 (m, 1 H), 2.16 (t, J = 8.4 Hz, 1
H), 1.90 (m, 2 H), 1.41 (s, 3 H), 1.28 (s, 3 H), 1.12 (d, J = 11.2 Hz,
1 H), 0.84 (s, 3 H); 13C NMR (75 MHz, CDCl3) d = 157.0, 156.6,
152.7, 145.9, 139.7, 134.6, 130.5, 129.9, 124.5, 124.2, 121.7, 119.6,
115.6, 101.4, 85.9, 77.9, 69.9, 51.3, 39.5, 38.1, 35.4, 28.6, 27.1,
26.4, 23.9; IR (KBr) 2918, 2870, 1526, 1429, 1410, 1369, 1227,
839 cm-1; MS(ESI) m/z 490 [M+MeOH]+; Analysis calculated for
C27H29BClN3O3: C, 66.21; H, 5.97; N 8.58. Found: C, 66.11; H,
6.12; N, 8.53.
for 1 h. The acetonitrile phase was separated and concentrated.
The residue was purified by silica gel column chromatography
with dichloromethane/ethanol (30/1) to give 7b (18.9 mg, 18%)
as a yellow solid: H NMR (400 MHz, CD3OD) d = 8.37 (s, 1
1
H), 7.86 (t, J =2.0Hz, 1 H), 7.66 (d, J = 2.4 Hz, 1 H), 7.57–
7.63 (m, 2 H), 7.43 (dd, J = 9.2, 2.4 Hz, 1 H), 7.25 (t, J =
8.0 Hz, 1 H), 7.23 (bs, 1 H), 7.04 (d, J = 9.2 Hz, 1 H), 6.61
(dt, J = 16.0, 4.4 Hz, 1 H), 5.91 (d, J =16.0 Hz, 1 H), 4.69 (d, J =
4.4 Hz, 2 H);13C NMR (75 MHz, CD3OD) d = 159.3, 159.0, 153.5,
146.2, 142.0, 135.5, 131.2, 129.9, 126.5, 125.5, 123.9, 122.1, 117.6,
104.3, 71.5; IR (KBr) 2926, 2855, 1597, 1560, 1526, 1508, 1364,
1227, 1063, 995, 835 cm-1; Analysis calculated for C17H15BClN3O3:
C, 57.42; H, 4.25; N, 11.82. Found: C, 57.45; H, 4.35;
N 11.76.
4-(3-Chloroanilino)-6-(2,9,9-trimethyl-3,5-dioxa-4-
boratricyclo[6.1.1.02,6]dec-4-ylmethoxy)quinazoline (6c)
[4-(3-Chloroanilino)quinazolin-6-yloxy]methylboronic acid (7c)
Synthesized from 5 (271 mg, 1.0 mmol) and 10 (286 mg, 1.0 mmol)
using the procedure described for 6a, to give 6c (91 mg, 19%) as a
yellow solid: 1H NMR (400 MHz, CDCl3) d = 8.73 (s, 1 H), 7.95
(t, J = 2.0 Hz, 1 H), 7.86 (d, J = 8.8 Hz, 1 H), 7.59 (dd, J = 9.2,
1.2 Hz, 1 H), 7.55 (dd, J = 9.2, 2.4 Hz, 1 H), 7.41 (bs, 1 H), 7.32
(t, J = 8.0 Hz, 1 H), 7.21 (d, J = 2.4 Hz, 1 H), 7.12 (dt, J = 8.0,
1.2 Hz, 1 H), 4.42 (d, J = 8.0 Hz, 1 H), 4.01 (s, 2 H), 2.38 (m, 1 H),
2.24 (m, 1 H), 2.12 (t, J = 8.4 Hz, 1 H), 1.92 (m, 2 H), 1.46 (s, 3 H),
1.30 (s, 1 H), 1.19 (d, J = 12.0 Hz, 1 H), 0.86 (s, 3 H);13C NMR
(75 MHz, CDCl3) d = 158.8, 156.4, 152.6, 145.6, 139.8, 134.7,
130.5, 129.9, 124.1, 124.0, 121.3, 119.2, 115.5, 100.4, 87.1, 78.6,
51.2, 39.5, 38.2, 35.2, 28.6, 27.0, 26.6, 23.9; IR (KBr) 2926, 2870,
1579, 1560, 1521, 1435, 1209, 1128, 1015, 841 cm-1; MS(ESI) m/z
486 [M+Na]+; Analysis calculated for C25H27BClN3O3: C, 64.75;
H, 5.87; N, 9.06. Found: C, 64.88; H, 5.80; N, 9.13.
Synthesized from 6c (136 mg, 0.24 mmol) and KHF2 (137 mg,
1.75 mmol) using the procedure described for 7b, to give 7c
(19.8 mg, 19%) as a yellow solid:1H NMR (400 MHz, CD3OD)
d=8.48 (s, 1 H), 7.88 (bs, 1 H), 7.72 (bs, 1 H), 7.53–7.62 (m, 2
H), 7.47 (d, J = 9.2 Hz, 1 H), 7.27 (t, J = 8.0 Hz, 1 H), 7.07
(d, J = 8.0 Hz, 1 H), 3.35 (s, 2 H);13C NMR (75 MHz, CD3OD)
d = 159.1, 155.6, 152.3, 146.5, 142.5, 135.3, 131.0, 129.4, 125.1,
125.0, 124.4, 122.0, 120.3, 118.8, 55.6; IR (KBr) 2924, 2852, 1558,
1541, 1522, 1508, 1387, 1340, 1225 cm-1; Analysis calculated for
C15H13BClN3O3: C, 54.67; H, 3.98; N, 12.75. Found: C, 54.55; H,
3.70; N, 12.91.
4-[4-(3-Chloroanilino)quinazolin-6-yloxymethyl]phenylboronic
acid (7d)
Synthesized from 6d (15 mg, 32 mmol) and KHF2 17.5 mg,
0.22 mmol) using the procedure described for 7b, to give7d (3.0 mg,
28%) as a yellow solid:1H NMR (400 MHz, CD3OD) d = 8.43 (s,
1 H), 7.85 (dd, J = 7.2, 2.4 Hz, 2 H), 7.50–7.67 (m, 5 H), 7.39
(m, 2 H), 7.27 (t, J = 8.0 Hz, 1 H), 7.07 (d, J = 8.0 Hz, 1 H),
5.16 (s, 2 H);13C NMR (75 MHz, CD3OD) d =159.6, 159.5, 153.0,
144.2, 141.6, 135.5, 153.2, 131.3, 128.7, 128.1, 127.4, 127.1, 125.9,
124.1, 122.4, 117.4, 104.7, 72.0; IR (KBr) 2903, 2856, 1579, 1570,
1528, 1508, 1380, 1251, 1016, 833cm-1; Analysis calculated for
C21H17BClN3O3: C, 62.18; H, 4.22; N, 10.36. Found: C, 62.46; H,
4.38; N, 10.40.
4-(3-Chloroanilino)-6-[4-(2,9,9-trimethyl-3,5-dioxa-4-
boratricyclo[6.1.1.02,6]dec-4-yl)-benzyloxy]quinazoline (6d)
Synthesized from 5 (271 mg, 1.0 mmol) and 13 (349 mg, 1.0 mmol)
using the procedure described for 6a, to give 6d (297 mg, 52%) as
a yellow solid:1H NMR (400 MHz, CDCl3) d = 8.73 (s, 1 H), 7.93
(t, J = 2.0 Hz, 1 H), 7.87–7.90 (m, 1 H), 7.75–7.83 (m, 1 H), 7.55
(m, 2 H), 7.49 (d, J = 8.0 Hz, 2 H), 7.34 (t, J = 8.0 Hz, 1 H),
7.15 (s, 1 H), 7.14 (d, J = 8.0 Hz, 1 H), 5.25 (s, 2 H), 4.47 (d, J =
7.2 Hz, 1 H), 2.44 (m, 1 H), 2.24 (m, 1 H), 2.16 (t, J = 5.2 Hz, 1
H), 1.92 (m, 2 H), 1.50 (s, 3 H), 1.32 (s, 3 H), 1.20 (d, J = 10.8 Hz,
1 H), 0.89 (s, 3 H);13C NMR (75 MHz, CDCl3) d = 157.1, 156.4,
152.8, 145.7, 139.6, 139.0, 135.3, 134.7, 130.7, 129.9, 126.6, 124.5,
124.2, 121.5, 119.4, 115.5, 101.3, 86.4, 78.3, 51.4, 39.5, 38.2, 35.5,
28.6, 27.0, 26.4, 24.0; IR (KBr)2918, 2870, 1570, 1528, 1404, 1364,
1229, 1092, 1020, 837 cm-1; MS(ESI) m/z 540 [M + H]+; Analysis
calculated for C31H31BClN3O3: C, 68.97; H, 5.79; N, 7.78. Found:
C, 68.91; H, 5.59; N, 7.78.
Kinase assays
The kinase activity of various tyrosine kinases was determined
by ELISA.38 EIA/RIA StripwellTM plates (Corning) were coated
by incubation overnight at 4 ◦C with 100 ml/well of 50 mg/ml
poly(Glu:Tyr, 4:1) peptide (Sigma) in PBS. The kinase reaction
was performed in the plates by addition of 50 ml of kinase
buffer (50 mM HEPES, 125 mM NaCl, 10 mM MgCl2, pH 7.4)
containing ATP (10 mM), 10 ng of recombinant EGFR, HER2,
Flt-1 or KDR (Invitrogen, catalytic domain), and inhibitors. After
20 min, the plates were washed three times with wash buffer (0.1%
Tween 20 in PBS) and incubated for 20 min with 50 ml/well
of 0.2 mg/ml HRP conjugated anti-phosphotyrosine antibody
(Santa Cruz). After two washes, the plates were developed by
addition of 50 ml/well tetramethylbenzidine (Sigma) and stopped
by addition of 50 ml/well of 2 N H2SO4. The absorbance at 450 nm
4-[4-(3-Chloroanilino)quinazolin-6-yloxy]but-2-enylboronic
acid (7b)
To a solution of 6b (142 mg, 0.29 mmol) in methanol (10 ml)
was added KHF2 (158 mg, 2.0 mmol) in water (10 ml), and the
reaction mixture was stirred at room temperature for 2 h. The
solvent was removed and the residue was dissolved in acetonitrile
(5 ml) and hexane (9 ml). Phenyl boronic acid (73 mg, 0.6 mmol)
was added and then the mixture was stirred at room temperature
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was measured by a 96-well plate reader (Tecan). Preincubation-
time dependent inhibition assay was generated based on previous
methods.45 The recombinant EGFR (10 ng) was incubated in
kinase assay buffer containing various concentrations of drug
except ATP. After incubation for 3, 5, or 15 min, the reaction was
initiated by addition of ATP (10 mM) into the reaction mixture.
Other procedures were performed as described above.
Detection of apoptosis
Phosphatidylserine externalization was measured using an An-
nexin V-FITC kit (Beckman Coulter) according to the manufac-
turer’s instructions. After incubation of A431 cells with the drugs,
the cells were suspended in 100 ml of 1X binding buffer, and added
5 ml of annexin V-FITC (5 mg/ml) and 2.5 ml of PI (250 mg/ml in
PBS). The cell suspensions were incubated for 10 min at 4 ◦C and
analyzed by a flow cytometer.
Cell growth assays
The human carcinoma cell lines, A431 (epidermoid carcinoma),
HepG2 (liver carcinoma), KatoIII (gastric carcinoma), and
SKBR3 (breast carcinoma) were used for the cell growth
assay. Each cell line (5 ¥ 103 cells/well of 96-well plate)
was incubated at 37 ◦C for 72 h in 100 ml of RPMI-1640
medium containing various concentrations of the boronic acid-
conjugated 4-anilinoquinazolines. After the incubation, 10 ml
of 3¢-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT, Sigma) in PBS (5 mg/ml) was added into the each well,
and the cells were further incubated at 37 ◦C for 4 h. After
the removal of the medium, 100 ml of DMSO was added, and
the absorbance at 570 nm was determined. For cell-based wash
experiment,15 A431 cells were incubated for 18 h with compounds
and then washed with fresh medium. After further incubation for
54 h, the rates of viable cells were determined by MTT assay. The
drug concentrations required to inhibit cell growth by 50% (GI50)
were determined from semilogarithmic dose–response plots.
Molecular docking and quantum mechanical simulations:
Docking and binding energy calculations were carried out based
on the X-ray structure of EGFR kinase domain in complex
with Tarceva (PDB code: 1M17).22 Based on this structure, the
entire kinase domain of the receptor protein (residues 672–964),
inhibitor, and a water molecule were selected for simulations. All
water molecules in the crystal structure were removed except
one molecule which mediate hydrogen bond network between
the protein and inhibitor (Fig. 8). Hydrogen atoms were added
to the protein, considering the terminal amino acid and all
charged amino acid residues the charged state. The positions
of added hydrogen atoms were minimized using MMFF94x
force field using the Molecular Operating Environment (MOE)
software (Chemical Computing Group Inc.). The binding site of
the boron-conjugated 4-anilinoquinazoline 7d in EGFR kinase
domain was defined based on that of Tarceva in the 1M17 X-
ray structure. As the initial structure, the quinazoline ring of
compound 7d was superimposed on that of Tarceva retaining
hydrogen bond network with the EGFR. Docking structures were
calculated in two steps. First, systematic conformation search of
compound 7d in the EGFR kinase domain was performed for
three rotatable bonds (Fig. 7) using the MOE software. Then two
conformations were selected, in which boronic acid moiety was
located at the Asp800 (model 1) or Cys797 (model 2 and model
3) interaction sites, followed by the MMFF94x minimization of
the inhibitor structure at the EGFR binding site. Second, the
detailed docking geometries for both Tarceva and compound
7d were further optimized by the hybrid QM/MM simulation
of quantum mechanics (QM) and molecular mechanics (MM)
methods. The QM/MM calculations were carried out employing
the ONIOM scheme29,30 as implemented in GAUSSIAN0346 using
the universal force field (UFF)47 for the MM region. The Hartree–
Fock (HF) method using the 6-31G(d) basis set was chosen as
the high level of theory for the QM region. The QM region
consisted of side chains of Cys775, Thr790, Cys797, and Asp800,
backbone of Met793, single water molecule, and the inhibitor
(Fig. 8). Both side chains and backbones were capped at the Ca
positions by using hydrogen link atoms, and the thiol group of
Cys797 was either the –SH or the –S- form. Other residues in
EGFR were treated with the UFF. With these optimized docking
geometries, the binding energies between EGFR and inhibitors
were calculated using the ab initio fragment molecular orbital
(FMO) method at the HF/6-31G(d) level of theory. The FMO
method is a fully QM approach and details of this method have
been described elsewhere.31–34 The approximations of electrostatic
potentials considered as the Mulliken orbital charge (esp-aoc) and
the fractional point charge (esp-ptc) were carried out when the
distance between the closest contact atoms in the two fragments
Immunoblot analysis
After stimulation for the specified period, the cells were washed
three times with PBS, dipped in 100 ml of ice-cold lysis buffer
(20 mM HEPES, pH 7.4, 1% Triton-X 100, 10% glycerol, 1 mM
sodium vanadate, 5 mg/ml of leupeptin, and 1 mM EDTA)
for 15 min, and disrupted with a Handy Sonic Disrupter, and
the lysate was boiled for 5 min in a sample buffer (50 mM
Tris, pH 7.4, 4% sodium dodecylsufate (SDS), 10% glycerol,
4% 2-mercaptoethanol, and 0.05 mg/ml of bromophenol blue)
at a ratio of 4:1. The proteins were separated by 10% SDS-
polyacrylamide gel electrophoresis and transferred onto a PVDF
membrane (Millipore). For the immunoblotting, antibodies used
were phosphor-EGFR(Tyr1173) antibody (Santa Cruz), EGFR-
antibody (Santa Cruz), phospho-Akt (Ser473) antibody (Cell
Signaling, Beverly, MA), and phospho-p44/42 MAP kinase
(Tyr204) antibody (Santa Cruz). After incubation with HRP-
conjugated secondary antibody (Santa Cruz), the blot was reacted
with ECL kit (GE healthcare), and the levels of each protein were
visualized by a ChemiDoc XRS image analyzer (Bio-Rad).
Cell cycle analysis
After incubation of A431 cells with the drugs, the cells were washed
◦
with PBS, and fixed with 70% ethanol for 2 h at 4 C. The cells
were incubated for 30 min at 37 ◦C in 1 ml of RNase solution
(0.25 mg/ml in PBS), and further incubated for 30 min at 4 ◦C in
propidium iodide staining solution (PI, 0.05 mg/ml in PBS). The
suspension was then passed through a 40 mm nylon mesh filter
and analyzed by a Cytomics FC500 flow cytometer (Beckman
Coulter).
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exceed 0.0 and 2.0 in the units of van der Waals radii (vdW),
respectively. In addition, the Coulomb interaction approximation
(dimmer-es) was applied for the fragment pair calculations in two
fragments with separations exceeding 2.0 in the same vdW unit.
The fragmentation of the system was as follows: each amino acid
residue of EGFR, the inhibitor molecule, and the water molecule
were treated as a single fragment. All the FMO calculations
were performed with the ABINIT-MP program (available at
http://www.ciss.iis.u-tokyo.ac.jp/rss21/).
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Mochizuki and Dr. Tatsuya Nakano for discussions on quantum
mechanical calculations. This work was supported by a Grant-in-
Aid for Science Research (B) (No. 18350090) from the Ministry
of Education, Culture, Sports, Science and Technology and the
“Core Research for Evolutional Science and Technology” project
of the Japan Science and Technology Agency (JST-CREST).
Provision of computational resources from the “Revolutionary
Simulation Software (RSS21)” project supported by the Ministry
of Education, Culture, Sports, Science, and Technology (MEXT),
Japan is gratefully acknowledged.
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