Anilinodialkoxyquinazolines: Screening epidermal growth factor receptor tyrosine kinase inhibitors for potential tumor imaging probes

Article abstract of DOI:10.1021/jm050607w

The epidermal growth factor receptor (EGFR), a long-standing drug development target, is also a desirable target for imaging. Sixteen dialkoxyquinazoline analogues, suitable for labeling with positron-emitting isotopes, have been synthesized and evaluated in a battery of in vitro assays to ascertain their chemical and biological properties. These characteristics provided the basis for the adoption of a selection schema to identify lead molecules for labeling and in vivo evaluation. A new EGFR tyrosine kinase radiometric binding assay revealed that all of the compounds possessed suitable affinity (IC₅₀ = 0.4-51 nM) for the EGFR tyrosine kinase. All of the analogues inhibited ligand-induced EGFR tyrosine phosphorylation (IC ₅₀ = 0.8-20 nM). The HPLC-estimated octanol/water partition coefficients ranged from 2 to 5.5. Four compounds, 4-(2′-fluoroanilino)- and 4-(3′-fluoroanilino)-6,7-diethoxyquinazoline as well as 4-(3′-chloroanilino)- and 4-(3′-bromoanilino)-6,7- dimethoxyquinazoline, possess the best combination of characteristics that warrant radioisotope labeling and further evaluation in tumor-bearing mice.

Full text of DOI:10.1021/jm050607w

J. Med. Chem. 2005, 48, 7445-7456
7445
Anilinodialkoxyquinazolines: Screening Epidermal Growth Factor Receptor
Tyrosine Kinase Inhibitors for Potential Tumor Imaging Probes
,
†
†,‡,§
|,⊥
†
†,#
Henry F. VanBrocklin,* John K. Lim,
Stephanie L. Coffing, Darren L. Hom, Kitaw Negash,
| |
†
|
Michele Y. Ono, Jennifer L. Gilmore, Ianthe Bryant, and David J. Riese II
Department of Functional Imaging, Lawrence Berkeley National Laboratory, Berkeley, California 94720-8119,
Department of Radiology, University of California, San Francisco, California 94143, and Department of Medicinal Chemistry
and Molecular Pharmacology, Purdue University, 1333 Heine Pharmacy Building, West Lafayette, Indiana 47907
Received June 25, 2005
The epidermal growth factor receptor (EGFR), a long-standing drug development target, is
also a desirable target for imaging. Sixteen dialkoxyquinazoline analogues, suitable for labeling
with positron-emitting isotopes, have been synthesized and evaluated in a battery of in vitro
assays to ascertain their chemical and biological properties. These characteristics provided
the basis for the adoption of a selection schema to identify lead molecules for labeling and in
vivo evaluation. A new EGFR tyrosine kinase radiometric binding assay revealed that all of
the compounds possessed suitable affinity (IC50 ) 0.4-51 nM) for the EGFR tyrosine kinase.
All of the analogues inhibited ligand-induced EGFR tyrosine phosphorylation (IC50 ) 0.8-20
nM). The HPLC-estimated octanol/water partition coefficients ranged from 2 to 5.5. Four
compounds, 4-(2′-fluoroanilino)- and 4-(3′-fluoroanilino)-6,7-diethoxyquinazoline as well as 4-(3′-
chloroanilino)- and 4-(3’-bromoanilino)-6,7-dimethoxyquinazoline, possess the best combination
of characteristics that warrant radioisotope labeling and further evaluation in tumor-bearing
mice.
Introduction
receptor family members has been shown to be a key
determinant in tumor growth and proliferation.6
,7
Protein tyrosine kinases (PTKs), enzymes that phos-
phorylate tyrosine residues on functional proteins, are
common mediators of signals that regulate many cel-
lular processes. PTKs can be divided into two subgroups
on the basis of their structural characteristics, nonre-
Overexpression of EGFR has been found in head and
neck tumors, gliomas, non-small-cell lung carcinoma
and tumors of the breast, ovaries, cervix, esophagus,
bladder, prostate, and kidney.7 There is extensive
literature on the clinical significance of increased EGFR
expression and signaling in tumors and the relationship
to other prognostic factors.8 In a majority of tumors,
poor survival rates correlate with EGFR overexpression.
For example, EGFR overexpression has been detected
in nearly 45% of the breast tumors studied10 and
receptor overexpression is inversely correlated with
ceptor cytoplasmic PTKs and receptor tyrosine kinases
1
(
RTKs). To date at least 20 families of receptor tyrosine
,9
kinases that share structural, most notably an intrinsic
tyrosine kinase domain, and functional similarities have
2,3
been classified. The epidermal growth factor receptor
EGFR) was one of the first oncogenes and receptor
(
4
,5
tyrosine kinases to be discovered. EGFR belongs to
the ErbB family of receptors, which also includes ErbB2
9
patient survival. EGFR overexpression is also inversely
correlated with estrogen receptor status, consistent with
(
HER2/Neu), ErbB3, and ErbB4. These receptors are
the failure of EGFR-positive patients to respond to
overexpressed in a variety of tumors.
hormonal therapies.1
1,12
The relevance of EGFR expres-
EGFR tyrosine kinase phosphorylation is stimulated
by epidermal growth factor (EGF) or transforming
growth factor R (TGFR) binding to the extracellular
ligand binding domain of the EGFR and subsequent
receptor dimerization. Signal transduction initiated by
these events regulates cellular proliferation, differentia-
tion, motility, adhesion, and apoptosis. These signaling
processes play an important role in normal epithelial
and stromal cell morphology; moreover, overexpression
or aberrant signaling from EGFR and the other ErbB
sion in tumors to prognostic outcome supports the years
of investment toward finding an EGFR-targeted thera-
peutic1
3-18
and the need for a diagnostic imaging agent.
Development of an imaging agent might also be a
valuable tool in the search for and the characterization
of EGFR-targeted therapeutics.
Two potential targets for EGFR-based probes are the
extracellular domain or the intracellular tyrosine kinase
domain. Radioactive probes that bind to the extracel-
lular domain of EGFR have been generated. EGF, the
5
3 amino acid, 6 kDa, natural ligand, binds to the EGFR
*
Corresponding author. Phone: 510.486.4083. Fax: 510.486.4768.
19
with a Kd of 0.1-1.0 nM and has been labeled directly
with iodine-123, iodine-125, and iodine-131. In vitro
E-mail: hfvanbrocklin@lbl.gov.
†
Lawrence Berkeley National Laboratory.
‡
University of California.
131
studies using [ I]EGF demonstrated its cytotoxic
§
Current address: Hitachi High Technologies America, Inc., San
potential.2
0 123
I-EGF has been used to image cervical
Jose, CA 95134.
|
21
Purdue University.
Current address: American Cyanamide Co., Agricultural Research
cancer in humans. Despite its ability to localize in
EGFR-rich tissue, radioiodinated EGF rapidly degrades
in vivo, releasing radioiodine, thereby reducing the
#
Division, Princeton, NJ 08543.
⊥
Current address: Pfizer Central Research, Groton, CT 06349.
1
0.1021/jm050607w CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/20/2005

7
446 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
VanBrocklin et al.
Figure 1. EGFR tyrosine kinase inhibitors13,16,34,55
lifetime of the label in the tumor.22 Iodinated dextran-
EGF conjugates increase retention of the iodine by
tumor cells but at the cost of higher molecular weight
rings (see Figure 1) of the anilino- or benzylamino-
125
quinazoline. The C ring substituents include 4-(3′-[ I]-
3
6,37
18
iodoanilino,
4-(3′-[ F]fluoro-5′-(trifluoromethyl)-
2
0
38
18
38
that affects tracer distribution. EGF has been labeled
with technetium-99m using a MAG3 or 2-iminothiolane
chelate and was found to accumulate in tumor xe-
nografts with more rapid clearance than labeled mono-
anilino), 4-(3′,4′-dichloro-6′-[ F]fluoroanilino), and
1
8
39
18
4-(3′-chloro-4′-[ F]fluoroanilino). The 7-[ F]fluoroet-
hoxy40 and the 6- or 7-[ C]methoxy
11
40,41
constitute the
A ring labeled substituents. Preliminary in vitro studies
2
3-25
125
clonal antibodies (Mabs).
Bifunctional chelation of
with the 3′-[ I]iodo analogue demonstrated receptor-
indium-111 to EGF using diethylenetriaminepentaacetic
acid (DTPA) has been shown to have cytotoxic effects
in vitro and is being investigated as a potential radio-
mediated uptake in cells containing high EGFR titer.37
A study of the carbon-11 methoxy derivative demon-
strated some uptake in human neuroblastoma xe-
2
6,27
111
42,43
therapeutic agent.
The imaging potential of In-
nografts in mice;
however, the 20-min half-life of the
EGF was found to be inferior to a labeled anti-EGFR
carbon-11 may not allow adequate time for the develop-
ment of good signal relative to background. Bonasera
and colleagues evaluated five fluorine-18-labeled com-
pounds.38 They studied the 4-(4′-[ F]fluoroanilino)-
1
11
antibody,
In-DTPA-Mab 528, in tumor-bearing
2
8
rats.
18
Several Mabs against EGFR have been developed as
antitumor agents. The radioiodinated Mab, EGFR1,
with high affinity for the EGFR has demonstrated good
18
dimethoxyquinazoline and the 4-(3′,4′-dichloro-6′-[ F]-
fluoroanilino)dimethoxyquinazoline in tumor-bearing
mice. These tracers did not accumulate in the tumors
nor was receptor-mediated uptake, based on blocking
studies, observed for the latter probe. Low receptor
affinity, high nonspecific binding, and probe metabolism
may have contributed to the inability of these com-
pounds to accumulate in tumor cells that overexpress
EGFR.
2
9
uptake in MCF-7 tumors grown in nude athymic mice
and good localization in human brain gliomas.30 Like-
wise, the indium-111-labeled C225 Mab has been tested
in patients with squamous cell lung carcinoma.31
A
direct-labeled technetium-99m anti-EGFR antibody, ior
egfr/r3, has demonstrated imaging sensitivity (84%),
specificity (100%), and accuracy (86.5%) in human
3
2
epithelial tumors. However, Mabs labeled with short-
lived PET isotopes, in general, have demonstrated
limited targeting success in large part due to pharma-
cokinetic constraints related mostly to their size.
To date, no small organic molecules with affinity for
the extracellular domain of the EGFR have been identi-
fied; however, a number of small molecules have been
shown to be potent (nano- to picomolar) inhibitors of the
intracellular EGFR tyrosine kinase at the ATP binding
site. A sampling of the different compound classes that
inhibit EGFR phosphorylation is shown in Figure 1. The
dialkoxyanilinoquinazolines (Figure 1) were chosen as
the lead compounds in the present study on the basis
Successful development of an imaging probe targeting
a new biomarker, in this case EGFR, requires an
adequate screening strategy for the selection of ligands
to be carried forward for labeling and, ultimately, in vivo
studies. It is neither economically nor logistically fea-
sible to label and evaluate every compound in animal
models. Likewise, navigating the structure-activity
relationships in the medicinal chemistry literature can
be challenging with respect to choosing an appropriate
imaging lead compound. For example, the fact that a
small molecule is a potent EGFR inhibitor does not
necessarily guarantee that it will possess desirable
EGFR imaging characteristics such as high receptor
affinity, high receptor selectivity, low nonspecific bind-
ing, rapid clearance, and suitable metabolism.
3
3-35
of the strong structure-activity data.
The development of imaging agents based on the
small-molecule EGFR inhibitors has been a recent area
of active research. A number of radiolabeled anilino-
quinazoline analogues have been reported. The com-
pounds incorporate labeled substituents on the A or C
In the current study, a small series of dialkox-
yquinazoline EGFR inhibitors suitable for labeling with
fluorine-18 (110-min half-life positron-emitter) or carbon-
11 (20-min half-life positron-emitter) has been synthe-

EGFR Inhibitors as Imaging Probes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7447
Scheme 1a
commercial supply was depleted, 4,5-diethoxybenzoic
acid 1 was nitrated to give 2 followed by reduction of
the nitro group to form the desired aminobenzoic acid
4
in moderate overall yield.
Preparation of the 4-chloroquinazolines followed a
previously described procedure (Scheme 2).33 Cyclization
of the dialkoxyanthranilic acids 4 or 5 with formamidine
hydrochloride at 210 °C gave the corresponding dialkox-
yquinazolinones, 6 or 7, in 55-65% yield. The quinazoli-
nones were subsequently refluxed with oxalyl chloride
in DMF and 1,2-dichloroethane to form the 4-chloro-
dialkoxyquinazolines 8 and 9 in good yield.
A modified coupling procedure was employed for the
production of the anilino- and (benzylamino)quinazo-
lines (Scheme 3). Anhydrous DMF was used as the
reaction solvent instead of the previously reported
a
Reagents and conditions: (i) 70% HNO3/glacial acetic acid/2
h/rt; (ii) SnCl2/HCl/2 h/rt; (iii) NaOH/reflux/1 h, then HCl.
Scheme 2a
2
-propanol. The reaction was carried out at 80 °C with
nearly quantitative conversion to the substituted ami-
noquinazoline hydrochloride (HCl) salt within 30 min
to 1 h depending on the aniline substituents. The
precipitated HCl salt was filtered from the DMF solu-
tion and was converted to the free base for semi-
preparative normal phase HPLC purification. The pure
quinazolines were reconverted to the more stable HCl
salt for the biological assays.
Chemical and physical data for all of the compounds
are presented in Table 1. All of the anilino- and
benzylquinazoline analogues were analyzed by analyti-
cal reversed-phase HPLC and found to be greater than
a
Reagents and conditions: (i) formamidine·HCl/heat then
NaOH/sonicate/1 h/rt; (ii) oxalyl chloride/DMF/1,2-dichloroethane/
reflux.
9
9% pure. Elemental analysis of all of the dialkox-
yquinazoline analogues in Table 1 agree with the
calculated values to within (0.4%
sized. Appropriate assays have been developed to de-
termine both functional and imaging characteristics,
including a new radiometric binding assay to measure
the affinity of the inhibitors for the enzyme. These
studies provide the basis needed for the selection of
ligands to be labeled and further evaluated as potential
imaging agents for the noninvasive determination of
EGF receptor density.
Lipophilicity Measurement. The lipophilicity of
compounds can affect their tissue permeability proper-
ties, which can impact their localization in target
tissues. Lipophilicity may also affect binding to low-
affinity nonspecific sites that can compromise target
tissue to background tissue ratios. The octanol/water
partition coefficients of the quinazoline compounds were
Results
44
estimated by a reversed-phase HPLC method. This
method has been previously used by us to determine
lipophilicities of steroid ligands.45 The estimated log Po/w
values are reported in Table 1. The lipophilicities
generally exhibited the expected trends with a couple
of noted exceptions. The lipophilicity was greater for the
diethoxy series relative to the corresponding dimethoxy
analogues. Within the series 10a-d and 11a-d, the
lipophilicity increased with increasing size of the halo-
gen from fluorine to iodine. Adding the trifluoromethyl
moiety to 10a and 11a increased the lipophilicity by
Chemistry. A small library of anilino- and benzyl-
dialkoxyquinazoline compounds, 10-17, was prepared
by coupling 4-chlorodialkoxyquinazoline 8 or 9 with the
appropriate substituted aniline or benzylamine (Scheme
3
). The 4-chloro-6,7-dimethoxyquinazoline was synthe-
sized as previously reported from the 4,5-dimethoxyan-
thranilic acid 5. As the 4-(3′-bromoanilino)-6,7-diethox-
33
3
3
yquinazoline was reported to possess more potent
biological activity than the corresponding 6,7-dimethoxy
analogue, we were interested in finding a convergent
synthetic route suitable for the preparation of several
diethoxy analogues. The previously reported two-step
conversion of the dimethoxybromoanilinoquinazoline
1.7-1.8 log units. Interestingly, adding an extra meth-
ylene to produce the benzylamine did not significantly
increase the compound lipophilicity (compare 14 and
1
6b, 15 and 17b). In contrast, the position of the
1
1c to the diethoxy analogue 10c (Scheme 3), using a
fluorine on the aniline (C ring, Figure 1) ring did have
a significant effect on the lipophilicity. The m- and
p-fluoroanilino analogues, 10a and 16b, had similar
lipophilicities, while the ortho-substituted analogue,
pyridinium hydrochloride melt to give the bishydroxy
intermediate followed by O-alkylation with iodoethane,
3
3
proceeded in a low 5.5% yield. This was inadequate
for the preparation of a series of the diethoxy analogues.
Thus, we produced the 4,5-diethoxyanthranilic acid 4
by two methods (Scheme 1). Initially, a small amount
of methyl 2-amino-4,5-diethoxybenzoate 3 was com-
mercially available from Aldrich Specialty Chemical.
The benzoate 3 was directly converted to 4 by saponi-
fication of the methyl ester. Alternatively, when the
1
6a, showed a 0.7 log unit decrease. This trend was
similar for the dimethoxy analogues.
Biological Activity. The 16 quinazoline compounds
were studied in a battery of in vitro assays to assess
their biological properties and to develop a basis for
screening these and future compounds for potential

7
448 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
VanBrocklin et al.
Scheme 3a
a
Reagents and conditions: (i) 3-haloaniline/DMF/heat; (ii) 3-fluoro-5-(trifluoromethyl)aniline/DMF/heat; (iii) 4-fluorobenzylamine/DMF/
heat; (iv) 2- or 4-fluoroaniline/DMF/heat.
Table 1. 4-Anilino- and 4-(Benzylamino)quinazolines Chemical and Physical Data
no.
type
R1
R2
R3
mp (°C)
formula
anal.
log Po/wa
1
0a
0b
0c
0d
1a
1b
1c
1d
2
A
A
A
A
A
A
A
A
A
A
B
B
C
C
C
C
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3
F
H
247-248
260-261
250-252
258-261
244.5-246
230-235
256-257.5
251-251.5
278-280
269-270.5
238.5-240
250-251
252-255
220.5-222
247-248
231-232
C18H18FN3O2·HCl
C18H18ClN3O2·HCl
C18H18BrN3O2
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
3.71 ( 0.12
4.31 ( 0.26
4.40 ( 0.25
4.62 ( 0.26
2.96 ( 0.10
3.51 ( 0.23
3.49 ( 0.22
3.65 ( 0.22
5.49 ( 0.29
4.66 ( 0.26
3.78 ( 0.23
3.02 ( 0.20
3.02 ( 0.11
3.73 ( 0.23
2.20 ( 0.11
2.87 ( 0.21
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Cl
Br
I
H
H
H
C18H18IN3O2·HCl
C16H14FN3O2·1.1HCl
C16H14ClN3O2·HCl
C16H14BrN3O2·HCl
C16H14IN3O2·HCl
C19H17F4N3O2·HCl
C17H13F4N3O2·HCl
C19H20FN3O2
C17H16FN3O2·HCl
C18H18FN3O2·1.4HCl
C18H18FN3O2·HCl
C16H14FN3O2·HCl
C16H14FN3O2·HCl
F
H
CH3
Cl
Br
I
H
CH3
CH3
CH3CH2
CH3
CH3CH2
CH3
H
H
F
CF3
CF3
3
F
4
5
6a
6b
7a
7b
F
F
CH3CH2
CH3CH2
CH3
F
H
F
H
F
H
F
CH3
H
a
Estimated by the reversed-phase HPLC method of Minick et al.44
imaging agents. A radiometric binding assay was de-
veloped to determine the relative binding affinities of
these compounds for the ATP binding site in the
tyrosine kinase domain of the receptor. The ability of
these molecules to inhibit EGFR tyrosine phosphoryla-
tion was probed. The specificity of a small subset of
compounds was determined by assessing inhibition of
ErbB2 and ErbB4 receptor phosphorylation. Finally, the
ability of the ligand to inhibit cellular DNA synthesis,
in cells dependent and not dependent on EGF for cell
proliferation, was evaluated. This assay was performed
in an effort to find a test that would be amenable to
high-throughput screening, the results of which would
potentially correlate with receptor binding and ulti-
mately with the pharmacokinetic distribution of the
tracer in vivo.
Receptor binding is a key characteristic that these
molecules must possess to be suitable imaging agents.
A receptor binding assay using EGF receptors extracted
from the A431 human carcinoma cell membranes was

EGFR Inhibitors as Imaging Probes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7449
Table 2. Biochemical Data for the EGFR Tyrosine Kinase (tk)
Inhibitors
Table 3. Inhibition of DNA Synthesis (IC50, nM)
MCF-7/
receptor binding
no.
MCF-10Aa
MCF-7b
MCF-10A
(IC50, nM)
receptor phosphorylation (IC50, nM)
1
0a
99 ( 16
108 ( 8
78 ( 12
153 ( 25
59 ( 5
173 ( 24
211 ( 45
585 ( 108
>3000
1634 ( 100
188 ( 25
489 ( 87
141 ( 17
622 ( 69
179 ( 31
612 ( 134
1923 ( 456
1087 ( 402
1695 ( 169
1571 ( 263
1956 ( 842
1982 ( 387
1613 ( 123
2433 ( 61
ND
4717 ( 1014
1433 ( 88
>7000
1101 ( 656
7487 ( 505
1950 ( 260
3375 ( 686
19.5
10.1
21.7
10.3
33.2
11.5
7.6
no.
EGFR tk
EGFR tk
ErbB2 tk
ErbB4 tk
10b
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0c
0d
1a
1b
1c
1d
2
1
0a
0b
0c
0d
1a
1b
1c
1d
2
8.17 ( 1.57
0.38 ( 0.13
0.41 ( 0.09
0.64 ( 0.15
31.9 ( 7.00
1.26 ( 0.00
0.66 ( 0.12
1.05 ( 0.51
8.95 ( 3.26
20.0 ( 10.2
17.0 ( 5.0
47.7 ( 14.1
9.31 ( 1.19
16.0 ( 3.7
32.2 ( 7.4
51.0 ( 11.7
6.5 ( 2.1
1.2 ( 0.2
3.2 ( 0.8
4.6 ( 2.0
6.3 ( 2.0
0.8 ( 0.2
2.6 ( 0.5
6.4 ( 2.7
>50
19.1 ( 2.9
6.6 ( 1.6
10.9 ( 2.8
15.8 ( 2.2
23.1 ( 4.2
12.8 ( 3.5
19.1 ( 2.5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
215 ( 87
69 ( 10
50 ( 19
59 ( 29
4.2
143 ( 52
231 ( 92
49 ( 16
>100
3
2.9
7.6
4
5
6a
6b
7a
7b
3
7.8
12.0
10.9
5.5
4
5
6a
6b
7a
7b
a
EGF-dependent human mammary epithelial cell line. b EGF-
independent human mammary tumor cell line.
for EGFR appears to be high (Table 2). For the four
compounds tested (10c, 10d, 11c, and 14), the ErbB2
and ErbB4 tyrosine phosphorylation (kinase) IC50 values
were at least 1 order of magnitude greater than the
EGFR tyrosine phosphorylation (kinase) IC50 values. We
do recognize that this is a comparison of phosphoryla-
tion inhibition rather than binding.
developed and used to study the relative binding affinity
of these compounds to the tyrosine kinase domain.
Iodine-125-labeled 11d (specific activity 583-596 Ci/
3
6
mmol) was employed as the radioligand, and nonspe-
cific binding was determined by adding 11c, the bromo
analogue, to the assay. EGFR binding values, expressed
as an IC50, are shown in Table 2. All of the compounds
demonstrated high affinity for the receptor with IC50s
ranging from 0.4 to 51 nM. In all cases the diethoxy
analogues had relatively higher affinity for the receptor
than the corresponding dimethoxy derivatives. 10b-
The specificity of the molecules for EGFR was also
determined by examining their effect on DNA synthesis
by the EGF-dependent MCF-10A human mammary
epithelial cell line and the EGF-independent MCF-7
human mammary tumor cell line. These cell lines were
treated with various concentrations of several of the
compounds, and DNA synthesis was measured by
1
0d and 11b-11d, the m-chloro-, -bromo-, and -iodoa-
nilino analogues, exhibited the highest relative affinities
nanomolar to subnanomolar) for the tyrosine kinase
domain. Three fluoroanilino analogues, 10a, 12, and
6a, displayed relative affinities slightly less than 10
nM, generally considered the upper limit for imaging
(
3
assaying H-thymidine incorporation. These data were
used to calculate the DNA synthesis IC50 values for each
compound in the two cell lines, shown in Table 3. The
MCF7 DNA synthesis IC50 values for the compounds
were at least 1 order of magnitude higher than the
corresponding MCF10A DNA synthesis IC50 values.
Because the DNA synthesis of MCF10A cells is EGF-
dependent, these data suggest that the compounds
inhibit MCF10A DNA synthesis by inhibiting the EGFR
rather than some other target.
1
4
6,47
agents,
while the remaining analogues had relative
affinities greater than 16 nM.
While receptor binding is absolutely necessary for
localization of a potential imaging agent, the ability to
inhibit receptor function, in this case ligand-induced
receptor tyrosine autophosphorylation, may be un-
coupled from ligand binding. A series of assays were
designed to examine the correlation between receptor
binding and inhibition of receptor phosphorylation or
inhibition of EGF-dependent DNA synthesis. The inhi-
bition of ligand-induced EGFR tyrosine autophospho-
rylation is reported in Table 2. All of the compounds
were potent inhibitors of EGFR tyrosine phosphoryla-
tion (kinase activity) with the exception of the 4-[3′-
fluoro-5′-(trifluoromethyl)anilino]-6,7-diethoxyquinazo-
Discussion
There are four well-established criteria for the devel-
opment of disease-specific radioprobes that would be
sensitive to changes in binding site concentration.4
They are (i) to identify a binding site whose concentra-
tion changes as a function of a specific disease, (ii) to
design and to produce a radioprobe that selectively
targets the binding site, (iii) to evaluate sensitivity as
a function of altered binding site concentration, and (iv)
to evaluate sensitivity relative to the selected disease.
The EGF receptor overexpression in various tumors
satisfies the first criterion. Identifying lead molecules
to test as imaging probes and developing an underlying
selection process to identify future candidate molecules,
the subject of the present effort, begins to address the
second criterion.
6,47
2
line analogue 12. There was no correlation (r ) 0.02)
between binding affinity and inhibition of ligand-
induced EGFR tyrosine phosphorylation. Indeed, in
contrast to the results of the receptor binding assay, in
the EGFR tyrosine phosphorylation assays the diethoxy
analogues were not consistently better inhibitors than
the corresponding dimethoxy analogues.
Specificity for the EGF receptor versus other receptor
tyrosine kinases, especially ErbB2 and ErbB4, is an-
other desirable imaging characteristic. Thus, a small set
of compounds was evaluated for inhibition of ligand-
induced ErbB2 and ErbB4 tyrosine phosphorylation
Designing and producing an enzyme- or receptor-
binding radioprobe involves several steps, often an
iterative process, intended to obtain a thorough under-
standing of the biochemical and physiological behavior
(kinase activity). The specificity of the compounds tested

7
450 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
VanBrocklin et al.
of the probe to match against a set of desirable imaging
characteristics.46,47 A receptor-binding radiotracer should
meet the following criteria: (i) high affinity for the
desired enzyme or receptor, typically <10 nM; (ii)
appropriate lipophilicity (coupled to cell membrane or
blood-brain barrier penetration, typically 1.5-3.0); (iii)
high selectivity for the enzyme or receptor (e.g. low
affinity for receptors within the same family or similar
proteins, typically >10:1); (iv) suitable metabolic prop-
erties (labeled metabolites can alter the distribution
profile of the probe); and (v) rapid clearance from
nontarget tissues and the body (necessary for good
target-to-background ratio, typically >3:1, and lower
radiation dose to the subject). In vitro data for a series
of dialkoxyquinazoline EGFR-targeted compounds have
been gathered and used to select probes for labeling and
in vivo evaluation. These data were used to establish a
ligand selection process. The process is detailed in the
context of the following discussion.
inhibition constants, the following question was posed:
Could these values be used as a primary determinant
for potential imaging probes? All of the compounds
possessed suitable affinity for the EGFR tk (IC50 range
) 0.4-51 nM). Likewise, all of the compounds were
nanomolar inhibitors of ligand-induced EGFR tyrosine
phosphorylation (IC50 range ) 0.8-23.1 nM) in whole
cells. Yet, there was a complete lack of correlation
between the matched set of binding and phosphorylation
data. On the basis of this small set of compounds, the
phosphorylation data may not be used to predict recep-
tor affinity. This is likely due to the fact that inhibition
of ligand-induced receptor phosphorylation is not only
a function of receptor binding affinity but is also a
function of the ability to penetrate a cell and gain access
to the receptor. Thus, the receptor binding assay along
with a measure of cell penetration (inhibition of EGFR
dependent DNA synthesis, see below) will be integral
as an initial screen for tracers. Compounds 10a-d,
1
1b-d, 12, and 16a meet the generally accepted
The dialkoxyquinazolines (Figure 1) were chosen as
the lead compounds in the present study on the basis
minimum affinity for receptor-based imaging agents, 10
nM.
of the reported structure inhibition relationships.3
3-35
Fry and colleagues demonstrated that the quinazoline
backbone and the 6,7-dimethoxy moieties were neces-
sary for enhanced EGFR tyrosine kinase inhibition. The
A subsequent screen of selected lead compounds for
the inhibition of ligand-induced tyrosine phosphoryla-
tion will identify those molecules that readily access the
receptor. For example, compound 12 has a suitable
affinity for EGFR in vitro but displays minimal inhibi-
tion of ligand-induced receptor tyrosine phosphorylation.
Thus, compound 12 probably possesses limited penetra-
tion of cells and/or limited EGFR access. Therefore,
compound 12 is likely to be of limited value as a
potential EGFR-specific tumor imaging agent. Com-
pounds 10a-d, 11b-d, and 16a are potent inhibitors
of EGFR phosphorylation.
In cases where facilitated transport is nonexistent,
cell membrane permeability is a direct function of the
diffusion coefficient and compound lipophilicity. If the
partition coefficient value is too low, the compound will
not cross a cell membrane; if the value is too high,
hydrophobic interactions with lipids and proteins will
dominate, leading to high nonspecific binding, compro-
mising image contrast. It has been demonstrated for
radiotracers crossing the blood-brain barrier that a log
Po/w range of 1.5-3 is optimal.56 While this range works
equally well for nonbrain receptor-targeted tracers,
estrogen radioprobes with higher lipophilicity (up to log
P ) 4.5) have been successful tumor imaging agents.57
Therefore, the log Po/w range for the selection of the
EGFR probes in the current study has been set from
1.5 to 4. The HPLC-derived lipophilicities (log Po/w)
ranged from 2.2 to 5.5. Of those compounds previously
chosen, 10a, 11b-d, and 16a fall within this desired
range.
6
,7-diethoxy analogue 10c exhibited a 4-fold lower
inhibition IC50 value (6 pM vs 25 pM), and halogen
substitution at the 2′, 3′, 4′, and 5′ positions, even 3′,4′-
dibromination, of the anilino ring (“C” ring Figure 1)
was well-tolerated. The fluorobenzylamino analogue was
not previously studied, but on the basis of the radio-
chemical availability of this analogue, it was included
48
here. The unsubstituted benzyl compound was evalu-
ated as an inhibitor and found to have a 3-fold lower
inhibition IC50 value (10 nM) compared to the corre-
sponding unsubstituted anilino compound (29 nM).3
The 3-fluoro-5-(trifluoromethyl)aniline as well as 2-, 3-,
and 4-fluoroaniline can be synthesized with fluorine-
3,35
1
8, so the corresponding nonradioactive analogues were
4
9
added to the study. Inhibition data from the 4-[3′-
trifluoromethyl)anilino]-6,7-dimethoxyquinazoline (in-
(
hibition IC50 ) 0.24 nM) and the 3′-fluoroanilino ana-
logue (inhibition IC50 ) 3.8 nM) supported their inclusion.
The preparation of the analogues for this study was
straightforward. The 4-chloro-dimethoxy- and 4-chloro-
diethoxy-quinazoline intermediates were synthesized
and coupled with the appropriate aniline or benzyl-
amine to yield the desired anilino- or (benzylamino)-
quinazolines in good yield. The approach outlined herein
improved upon the chemistry previously described by
3
3
Bridges et al. and provides a suitable starting point
to generate libraries of anilino- or (benzylamino)-
quinazolines with any dialkoxy or mixed alkoxy sub-
stituents in the 6 and 7 positions.
Selectivity for the chosen receptor is the final in vitro
test. One preliminary measure of receptor selectivity
that was employed in this study was to evaluate ErbB2
and ErbB4 inhibition. All of the compounds were much
less potent inhibitors of ligand-induced ErbB2 and
ErbB4 phosphorylation than of ligand-induced EGFR
phosphorylation. Indeed, the compounds tested here
exhibit between 1 and 2 orders of magnitude of selectiv-
ity for the EGFR over ErbB2 or ErbB4. All of the
compounds tested may be reasonable EGFR-selective
tumor-imaging agents. The optimal agent would exhibit
A receptor binding assay was developed to study the
affinity of the compounds for the tyrosine kinase binding
site and determine the correlation, if any, between
binding and inhibition values. In vitro equilibrium
binding assays provide a good means of segregating
potential receptor-based imaging agents from those
whose unfavorable kinetics (high binding potential,
Bmax/Kd) might lead to flow-mediated distribution.47 As
all of the literature for the quinazolines and other
classes of compounds targeting the EGFR tk reported

EGFR Inhibitors as Imaging Probes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7451
the least amount of avidity for other RTKs. In many
types of tumor cells ErbB2 is frequently overexpressed
at levels comparable to the levels of EGFR overexpres-
but the tumor-to-blood ratio was only 0.6. At 60 min
the tumor uptake rose slightly to 1.34% ID/g with a
tumor-to-blood ratio of 1.62. This uptake may be as-
sociated with EGFR targeting, but receptor-blocking
studies were not performed to demonstrate receptor-
mediated uptake. Bone uptake, an indicator of metabolic
defluorination, was also not assessed. A second fluorine-
labeled analogue, 4-(3′,4′-dichloro-6’-fluoroanilino)-6,7-
dimethoxyquinazoline, was tested in tumor-bearing
mice. Tumor accumulation of the probe never exceeded
blood levels and the uptake was not receptor-mediated.
Several factors may have contributed to the limited
tumor accumulation in vivo, including high nonspecific
binding or diminished availability due to untoward
metabolism.
It is clear from the preliminary studies that develop-
ment of an effective EGFR imaging probe may present
some challenges. Thus, effective screening of candidate
probes and establishment of baseline data are essential
for a radiopharmaceutical development program target-
ing EGFR.
7
sion. Similarly, ErbB4 is expressed in the normal
59
mammary epithelium and infrequently overexpressed
5
8, 59
in tumor tissue.
These observations highlight the
need for EGFR-selective imaging agents that exhibit
minimal binding to ErbB2 and ErbB4, among other
RTKs, to determine and understand EGFR expression
using molecular imaging techniques.
The quinazoline compounds were also assayed for
inhibition of cellular DNA synthesis in EGF-dependent
(MCF10A) and EGF-independent (MCF7) cell lines. This
assay, like the inhibition of ligand-induced receptor
tyrosine phosphorylation assays, not only assesses the
specificity of a compound for EGFR but also provides a
measure of a compounds’ ability to penetrate cells and
specifically target the EGFR. Thus, it is not surprising
that compound 12, which we hypothesized failed to
inhibit ligand-induced EGFR tyrosine phosphorylation
because it exhibits limited cell penetration, also failed
to inhibit EGF-dependent and -independent DNA syn-
thesis.
Conclusion
In general, the MCF10A DNA synthesis IC50 values
are much lower than the MCF7 DNA synthesis IC50
values. This suggests that the compounds inhibit DNA
synthesis in the MCF10A cells by primarily targeting
EGFR. Indeed, this assay has been used to connote that
lavendustin A analogues, which inhibit EGFR kinase
activity, inhibit cell proliferation by inhibiting tubulin
polymerization.50 This indicates that at least some of
the compounds inhibit DNA synthesis in the MCF7 cells
by targeting a protein other than the EGFR. Compounds
that may target ATPases other than the EGFR would
be expected to exhibit reduced MCF7/MCF10A DNA
synthesis IC50 ratios. Compounds with reduced ratios
include 11d, 13, and 17b. Such molecules may not be
specific for the EGFR and may not be appropriate for
further investigation as potential EGFR-specific tumor
imaging agents. Compounds 10a-d, 11b,c, 14, 16a,b,
and 17a exhibit elevated MCF7/MCF10A DNA synthe-
sis IC50 ratios and by this criterion may be suitable for
further investigation as potential EGFR-specific tumor-
imaging agents.
Anilinodialkoxyquinazolines suitable for labeling with
radioisotopes were readily prepared using an approach
amenable to a synthetic library of aminoquinazoline
analogues. All of the dialkoxy analogues possessed
suitable affinity for the EGF receptor and all analogues
were potent inhibitors of ligand-induced EGFR phos-
phorylation. They exhibited a range of lipophilicities
based on the A ring and C ring substituents. Selectivity,
as determined by comparison of ErbB2 and ErbB4
receptor inhibition to EGFR inhibition, favored the
EGFR by 1-2 orders of magnitude. On the basis of
measures of affinity, lipophilicity, and selectivity, ana-
logues 10a, 11b, 11c, and 16a, were selected for further
evaluation as tumor imaging probes.
Experimental Section
Chemistry. Unless otherwise noted, all solvents and re-
agents were obtained from commercial suppliers (Aldrich
Chemical, Co., Lancaster Synthesis, Inc., VWR, etc.) and were
used without further purification. Melting points were deter-
mined using a Mel-Temp melting point apparatus and are
reported uncorrected. NMR spectra were recorded on either a
Bruker VBAMX 300 300 MHz or AMX 400 400 MHz. Chemical
shifts are reported in ppm (δ) relative to an internal standard.
Elemental analyses were performed by the Microanalytical
Laboratory in the College of Chemistry, University of Cali-
fornia, Berkeley. Mass spectral data was obtained on a Perkin-
Elmer SCIEX mass spectrometer at the SynPep Corp. facility
These criteria taken together support the further
investigation of compounds 10a, 11b, 11c, and 16a.
Compounds 10a and 16a, 4-(3′-fluoroanilino)- and 4-(2′-
fluoroanilino)-6,7-diethoxyquinazoline, respectively, may
both be labeled with the 110-min half-life fluorine-18.
Compound 11b, 4-(3′-chloroanilino)-6,7-dimethoxyquin-
azoline, may be labeled with the 20-min half-life carbon-
(
Dublin, CA) or on a VG ProSpec mass spectrometer at the
Mass Spectrometry Facility in the College of Chemistry,
1
1; however, as pointed out earlier, the short half-life
University of California, Berkeley.
may not afford the time necessary to achieve the desired
imaging pharmacokinetics. Compound 11c, 4-(3′-bro-
moaniline)-6,7-dimethoxyquinaxoline, may be a candi-
date for bromine-76 (16.2h positron emitter) labeling.
2-Nitro-4,5-diethoxybenzoic Acid (2). A flask immersed
in a room-temperature water bath was charged with 3,4-
diethoxybenzoic acid 1 (12.9 g, 61.4 mmol) and acetic acid
(
3
glacial, 52 mL). Over a 15-min period, HNO (70%, 54 mL)
was added dropwise with stirring. The deep orange solution
was stirred for an additional 125 min at room temperature.
The reaction was quenched upon addition of 110 g of ice. A
yellow precipitate formed that was filtered and washed with
3
8
Interestingly, Bonasera et al. have recently labeled
three of the analogues studied in this paper, 11a, 13,
and 17b, and injected one of them, 17b, into A431
tumor-bearing mice. Compound 13 was not studied in
vivo due to low measured EGFR inhibition values, and
compound 11a was not readily labeled with fluorine-
H
2
O (3 × 50 mL). The resulting yellow-white solid was
dissolved in ether (150 mL). The ether was washed with 1 N
NaOH (3 × 60 mL). The aqueous washings were combined and
acidified with concentrated HCl, resulting in the production
of a pale yellow precipitate. The precipitate was filtered,
1
8
1
8. Tumor uptake of [ F]17b at 30 min was greater
than 1% of the injected dose per gram (%ID/g) of tissue,
washed with H
2
O (3 × 100 mL), and dissolved in ether. The

7
452 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
VanBrocklin et al.
ether was dried over MgSO
0.55 g (67%) of 2: mp 142-145 °C; H NMR (CDCl
s, 1H, H-6), 7.21 (s, 1H, H-3), 4.20 (q, 2H, CH CH , J ) 6.9
Hz), 4.17 (q, 2H, CH CH , J ) 6.9 Hz), 1.50 (t, 6H, CH CH , J
7.1 Hz); EI MS [M+] 255 (100).
-Amino-4,5-diethoxybenzoic Acid (4). Method a. A
4
and concentrated in vacuo to yield
OCH
2
CH
3
); APCI MS [M + 1] 253.1 (100), 255.1 (33). Anal.
1
1
3
) δ 7.36
2 2
(C12H13ClN O ) C, H, N.
(
2
3
4-Chloro-6,7-dimethoxyquinazoline (9). 6,7-Dimethox-
yquinazolin-4-one 7 (3.40 g, 16.5 mmol) was converted to 9 in
2
3
2
3
)
52% yield (1.91 g) following the procedure for compound 8: mp
1
2
184-186 °C; H NMR (CDCl
3
) δ 8.86 (s, 1H, ArH), 7.38 (s, 1H,
round-bottom flask containing 2-amino-4,5-diethoxy-methyl
benzoate 3 (1.00 g, 4.19 mmol, Sigma-Aldrich Rare Chemicals)
was mixed with 6.25 N NaOH (2.67 mL, 16.7 mmol) and water
ArH), 7.32 (s, 1H, ArH), 4.05 (s, 6H, OCH ); APCI MS [M + 1]
2 2
225.1 (100), 227.1 (30). Anal. (C10 ClN O ) C, H, N.
3
H
9
4-(3′-Chloroanilino)-6,7-diethoxyquinazoline Hydro-
chloride (10b). General Coupling Procedure. A solution
of 4-chloro-6,7-diethoxyquinazoline 8 (71 mg, 0.32 mmol) and
3-chloroaniline (40 µL, 0.38 mmol) in 3 mL of DMF was heated
at 80 °C under argon for 40 min. The reaction was cooled at
room temperature for 1 h. Ethyl acetate (2 mL) was added.
The resulting precipitate was filtered and further washed with
20 mL of ethyl acetate to give the HCl salt 10a (100 mg, 90%).
The salt was converted to the free base by dissolving 10b in a
mixture of 3 mL of ethyl acetate and 3 mL of 1 N NaOH. The
biphasic mixture was stirred vigorously for several minutes.
The ethyl acetate layer was filtered, washed with water (3 ×
(
5.0 mL). The solution was refluxed for 1 h to give a clear
brown solution. After cooling at ambient temperature for 10
min, water (15 mL) was added to the flask and the solution
was titrated to pH 6 with 1 N HCl. The solution was further
cooled in an ice bath for 30 min. The precipitate was filtered,
washed with water (100 mL) and pentanes (50 mL), and dried
in vacuo over P
,5-diethoxybenzoic acid as a pale yellow solid.
Method b. An oven-dried round-bottomed flask (250 mL)
equipped with a stirring bar was immersed in a room-
temperature water bath and charged with SnCl ·2H O (24.9
2 5
O overnight to give 0.83 g (76%) of 2-amino-
4
2
2
g, 110 mmol) and HCl (concentrated, 100 mL). 2-Nitro-4,5-
diethoxybenzoic acid 2 (1.64 g, 6.43 mmol) was added and the
mixture was stirred for 120 min at room temperature. The
mixture was diluted with HCl (concentrated, 40 mL), filtered,
and washed with concentrated HCl. The resulting white solid
4
1 mL), and dried over MgSO . The ethyl acetate was filtered
and the volume was reduced to less than 1 mL. The solution
was applied to a normal phase semipreparative HPLC (What-
man M9/50 partisil 10 column, 70:30 EtOAc: hexane, 6 mL/
min., UV 254 nm) for purification. The fraction containing the
free base was concentrated to dryness and the residue was
dissolved in MeOH (8 mL) with gentle heating. HCl (1 N, 3
mL) was added and the solution was placed in an ice bath.
The precipitate was filtered and washed with ethyl acetate to
give 72 mg (65%) of the 4-(3-chloroanilino)-6,7-diethyox-
was taken up in H
remaining undissolved material. The pH of the filtrate was
adjusted to 4.5 using NH OH. The deep purple solution was
extracted with CH Cl
(3 × 300 mL). The organic washings
were combined, dried over MgSO , and concentrated in vacuo,
to yield 0.64 g (44%) of a purple-white powder
2
O (500 mL) and filtered to remove the
4
2
2
4
1
yquinazoline hydrochloride salt 10b: mp 260-261 °C; H NMR
1
(CD OD) δ 8.72 (s, 1H, H-2), 7.97 (s, 1H, Ar-H), 7.87 (s, 1H,
:
mp 120-126 °C; H NMR (CDCl
s, 1H, H-3), 4.06 (q, 2H, CH CH , J ) 6.8 Hz), 4.01 (q, 2H,
CH CH , J ) 6.8 Hz), 1.46 (t, 3H, CH CH , J ) 6.8 Hz), 1.41
t, 3H, CH CH , J ) 6.8 Hz); EI MS [M+] 225 (100).
3
) δ 7.37 (s, 1H, H-6), 6.11
3
(
2
3
H-2′), 7.65 (d, 1H, H-4′, J ) 8.0 Hz), 7.47 (t, 1H, H-5′ J ) 8.1
Hz), 7.35 (d, 1H, H-6′, J ) 8.0 Hz), 7.20 (s, 1H, Ar-H), 4.31 (q,
2
3
2
3
(
2
3
4H, OCH
2
CH
3
, J ) 6.9 Hz), 1.55 (t, 6H, OCH
2
CH
3
, J ) 7.0
Hz); EI MS [M+] 343 (100), 345 (34). Anal. (C18H18ClN O ) C,
3 2
6
,7-Diethoxyquinazolin-4-one (6). A modified literature
33
H, N.
procedure was developed to produce 6. A 500 mL round-
bottom flask equipped with an air condenser was charged with
-amino-4,5-diethoxybenzoic acid 4 (1.36 g, 6.04 mmol) and
4-(3′-Fluoroanilino)-6,7-diethoxyquinazoline Hydro-
chloride (10a). Similar treatment of 8 (0.021 g, 0.083 mmol)
2
formamidine hydrochloride (0.70 g, 8.76 mmol). The solids
were thoroughly mixed and then heated to 200 °C under an
argon atmosphere for 15 min. The heating block temperature
was adjusted to 80 °C and the solution cooled to 80 °C over 40
min. Dilute NaOH (0.33 N, 20 mL) was added to the flask.
The mixture was sonicated at room temperature for 1 h,
producing in a dark gray-purple suspension. The solid was
filtered and washed with water (200 mL), pentanes (200 mL),
and ethyl acetate (200 mL) to give 6,7-diethoxyquinazolin-4-
with 3-fluoroaniline yielded 10a (77%): mp 247.0-248.0 °C;
1
3
H NMR (CD OD) δ 8.73 (s, 1H, H-2), 7.99 (s, 1H, H-5), 7.66
(d, J ) 10.6 Hz, 1H, H-2′), 7.53 (m, 1H, H-5′), 7.53 (m, 1H,
H-6′), 7.21 (s, 1H, H-8), 7.08 (dd, J ) 9.1, 7.1 Hz, 1H, H-4′),
4.33 (q, J ) 6.9 Hz, 4H, CH
CH CH O), 1.54 (t, J ) 6.9 Hz, 3H, CH
327. Anal. (C18 ‚HCl) C, H, N.
3
CH
2
O), 1.55 (t, J ) 6.9 Hz, 3H,
3
2
3
CH O); EI MS [M]
2
H
3 2
18FN O
4-(3′-Bromoanilino)-6,7-diethoxyquinazoline Hydro-
chloride (10c). Similar treatment of 8 (59.5 mg, 0.24 mmol)
33
one as an off-white solid. Drying in vacuo overnight over P
2
O
5
with 3-bromoaniline yielded 10c (50%): mp 250-252 °C (lit.
1
1
gave 0.80 g (57%) of 6: mp 248-251.5 °C; H NMR (CD
δ 7.98 (s, 1H, ArH), 7.55 (s, 1H, ArH), 7.11 (s, 1H, ArH), 4.20
q, 2H, CH CH , J ) 6.8 Hz), 4.16 (q, 2H, CH CH , J ) 6.8
Hz), 1.48 (t, 3H, CH CH , J ) 6.8 Hz), 1.46 (t, 3H, CH CH , J
6.8 Hz); EI MS [M+] 234 (100).
3
OD)
3 2
mp 155-167 °C, free base); H NMR [(CD ) SO] δ 11.17 (br s,
1H, NH), 8.86 (s, 1H, H-2), 8.19 (s, 1H, Ar-H), 7.99 (s, 1H,
H-2′), 7.73 (d, 1H, H-4′, J ) 7.6 Hz), 7.51 (d, 1H, H-6′, J ) 7.5
Hz), 7.45 (t, 1H, H-5′ J ) 8.0 Hz), 7.23 (s, 1H, Ar-H), 4.27 (m,
(
2
3
2
3
2
3
2
3
)
4H, OCH
2
CH
3
, J ) 7.2 Hz), 1.45 (t, 6H, OCH
2 3
CH , J ) 6.8
6
,7-Dimethoxyquinazolin-4-one (7). 2-Amino-4,5-dimeth-
Hz); HREIMS calcd for C18H18BrN O
3 2
m/z (M+) 387.05824,
oxybenzoic acid 5 (5.0 g, 25.3 mmol) was converted to 7 in 65%
389.05619, found 387.05776, 389.05532. Anal. (C18
3 2
H18BrN O )
yield (3.64 g) following the procedure for compound 6: mp
C, H, N.
2
1
(
78-278.5 °C (lit.33 mp 295-298 °C); H NMR [(CD
1
3
)
2
SO] δ
4-(3′-Iodoanilino)-6,7-diethoxyquinazoline Hydrochlo-
ride (10d). Similar treatment of 8 (116.1 mg, 0.46 mmol) with
2.07 (br s, 1H, NH), 7.98 (s, 1H, ArH), 7.43 (s, 1H, ArH), 7.11
), 3.87 (s, 3H, OCH ).
-Chloro-6,7-diethoxyquinazoline (8). Following a modi-
1
s, 1H, ArH), 3.90 (s, 3H, OCH
3
3
3-iodoaniline yielded 10d (95%): mp 258.5-261 °C; H NMR
4
3 2
[(CD ) SO] δ 11.19 (br s, 1H, NH), 8.86 (s, 1H, H-2), 8.19 (s,
3
3
1H, Ar-H), 8.10 (s, 1H, H-2′), 7.75 (d, 1H, H-4′, J ) 8.0 Hz),
7.67 (d, 1H, H-6′, J ) 8.0 Hz), 7.30 (s, 1H, Ar-H), 7.29 (t, 1H,
fied literature procedure, DMF (0.94 mL, 12.1 mmol) was
added dropwise to a solution of 1,2-dichloroethane (8.1 mL)
and oxalyl chloride (1.1 mL, 12.6 mmol) stirring under argon,
vigorously releasing gas. Following cessation of the gas
production, 6,7-diethoxyquinazolin-4-one 6 (1.89 g, 8.1 mmol)
was added to the thick white slurry and then refluxed for 2.5
h, resulting in a yellow-brown suspension. The reaction was
H-5′ J ) 8.0 Hz), 4.27 (m, 4H, OCH
2
CH
, J ) 6.8 Hz). APCI MS [M+1] 436.0 (100). Anal.
3 2
H18IN O ·HCl) C, H, N.
3
), 1.44 (t, 6H,
OCH CH
2
3
(C18
4-(3′-Fluoroanilino)-6,7-dimethoxyquinazoline Hydro-
chloride (11a). Similar treatment of 9 (0.032 g, 0.14 mmol)
with 3-fluoroaniline yielded 11a (27%): mp 244.5-246.0 °C
2 4
quenched by addition of Na HPO (0.5 M, 16.8 mL) followed
3
3
1
by stirring in an ice bath for 1 h. The suspension was filtered
and washed with water (200 mL) to isolate 4-chloro-6,7-
3
(lit. mp 253-254 °C); H NMR (CD OD) δ 8.72 (s, 1H, H-2),
7.82 (s, 1H, H-5), 7.47 (d, J ) 10.6 Hz, 1H, H-2′), 7.33 (m, 1H,
H-5′), 7.33 (m, 1H, H-6′), 7.03 (s, 1H, H-8), 6.89 (dd, J ) 8.6,
diethyoxyquinazoline 8 as a pale gray solid (1.48 g, 73%): mp
1
1
39-140 °C; H NMR (CDCl
3
) δ 8.82 (s, 1H, ArH), 7.35 (s, 1H,
7.3 Hz, 1H, H-4′), 3.88 (s, 6H, CH
·1.1HCl) C, H, N.
3
O); EI MS [M] 299. Anal.
ArH), 7.28 (s, 1H, ArH), 4.26 (m, 4H, OCH
2
CH
3
), 1.55 (m, 6H,
3 2
(C16H14FN O

EGFR Inhibitors as Imaging Probes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7453
4
-(3′-Chloroanilino)-6,7-dimethoxyquinazoline Hydro-
3H, CH
3
CH
2
O); HREIMS calcd for C18
H
18FN m/z (M+)
3
O
2
chloride (11b). Similar treatment of 9 (71.1 mg, 0.32 mmol)
327.13831, found 327.13779. Anal. (C18
H
18FN O ·1.4HCl) C,
3 2
33
with 3-chloroaniline yielded 11b (90%): mp 230-235 °C (lit.
H, N.
1
mp 261-262 °C); H NMR (CD
3
OD) δ 8.66 (s, 1H, H-2), 7.93
4-(4′-Fluoroanilino)-6,7-diethoxyquinazoline Hydrochlo-
(
s, 1H, Ar-H), 7.90 (s, 1H, H-2′), 7.66 (d, 1H, H-4′, J ) 8.0 Hz),
ride (16b). Similar treatment of 8 (0.10 g, 0.40 mmol) with
1
7
.44 (t, 1H, H-5′ J ) 8.1 Hz), 7.29 (d, 1H, H-6′, J ) 8.0 Hz),
.21 (s, 1H, Ar-H), 4.02 (s, 6H, OCH ); HREIMS calcd for
m/z (M+) 315.07745, 317.07450, found 315.07682,
17.07303. Anal. (C16 ·HCl) C, H, N.
-(3′-Bromoanilino)-6,7-dimethoxyquinazoline Hydro-
chloride (11c). Similar treatment of 9 (312 mg, 1.39 mmol)
4-fluoroaniline yielded 16b (97%): mp 252.0-255.0 °C;
H
7
3
3 2
NMR ((CD ) SO) δ 10.06 (s, 1H, NH), 8.77 (d, J ) 1.8 Hz, 1H,
C
16
H
14ClN
3
O
2
H-2), 8.27 (s, 1H, H-5), 7.68 (m, 2H, H-2′, H-6′), 7.32 (s, 1H,
H-8), 7.29 (dd, J ) 6.7, 6.6 Hz, 2H, H-3′, H-5′), 4.25 (q, J ) 5.1
3
3 2
H14ClN O
4
Hz, 2H, CH
(t, J ) 5.1 Hz, 3H, CH
3
CH
2
O), 4.20 (q, J ) 5.1 Hz, 2H, CH
3 2
CH O), 1.41
3
CH O), 1.40 (t, J ) 5.1 Hz, 3H, CH -
2
3
1
CH O); APCI MS [M + 1] 328.3. Anal. (C H FN O ·HCl) C,
with 3-bromoaniline yielded 11c (87%): mp 256-257.5 °C; H
2
18 18
3
2
H, N.
NMR [(CD
3 2
) SO] δ 11.35 (br s, 1H, NH), 8.88 (s, 1H, H-2), 8.29
(
7
7
s, 1H, Ar-H), 8.01 (s, 1H, H-2′), 7.77 (d, 1H, H-4′, J ) 8.0 Hz),
4-(2′-Fluoroanilino)-6,7-dimethoxyquinazoline Hydro-
.50 (d, 1H, H-6′, J ) 8.0 Hz), 7.45 (t, 1H, H-5′ J ) 8.0 Hz),
chloride (17a). Similar treatment of 9 (0.033 g, 0.15 mmol)
.32 (s, 1H, Ar-H), 4.00 (s, 3H, OCH
3
), 3.99 (s, 3H, OCH
3
); EI
with 2-fluoroaniline yielded 17a (50%): mp 231.0-232.0 °C;
1
MS [M+] 359 (100), 361 (97). Anal. (C16
H
14BrN
3
O
2
·HCl) C, H,
3
H NMR (CD OD) δ 8.65 (s, 1H, H-2), 7.97 (s, 1H, H-5), 7.58
N.
(ddd, J ) 7.7, 7.5 Hz, 1.10 Hz, 1H, H-5′), 7.44 (m, 1H, H-4′),
.33 (d, J ) 7.7 Hz, 1H, H-6′), 7.29 (m, 1H, H-3′), 7.25 (s, 1H,
H-8), 4.09 (s, 3H, CH O), 4.07 (s, 3H, CH O); HREIMS calcd
for C16 m/z (M+) 299.10701, found 299.10648. Anal.
(C16 14FN ‚HCl) C, H, N.
7
4
-(3′-Iodoanilino)-6,7-dimethoxyquinazoline Hydrochlo-
3
3
ride (11d). Similar treatment of 9 (276 mg, 1.23 mmol) with
3
3
3 2
H14FN O
3
2
1
-iodoaniline yielded 11d (92%): mp 251-251.5 °C (lit. mp
1
H
3 2
O
73 °C); H NMR [(CD
3 2
) SO] δ 11.33 (br s, 1H, NH), 8.90 (s,
4
-(4′-Fluoroanilino)-6,7-dimethoxyquinazoline Hydro-
H, H-2), 8.30 (s, 1H, Ar-H), 8.28 (s, 1H, H-2′), 7.79 (d, 1H,
chloride (17b). Similar treatment of 9 (0.10 g, 0.45 mmol)
H-4′, J ) 8.0 Hz), 7.69 (d, 1H, H-6′, J ) 8.0 Hz), 7.35 (s, 1H,
Ar-H), 7.31 (t, 1H, H-5′ J ) 8.0 Hz), 4.04 (s, 3H, OCH ), 4.02
s, 3H, OCH ); APCI MS [M + 1] 408.0 (100). Anal. (C16
IN ·HCl) C, H, N.
-[3′-Fluoro-5′-(trifluoromethyl)anilino)]-6,7-diethoxy-
quinazoline Hydrochloride (12). Similar treatment of 8
58.5 mg, 0.23 mmol) with 3-fluoro-5-(trifluoromethyl)aniline
with 4-fluoroaniline yielded 17b (94%): mp 247.0-248.0 °C;
H NMR ((CD ) SO) δ 10.12 (br. s, 1H, NH), 8.79 (s, 1H, H-2),
3 2
3
1
(
3
14
H -
8
6
3
.25 (s, 1H, H-5), 7.68 (m, 2H, H-2′, H-6′), 7.30 (dd, J ) 7.0,
.2 Hz, 2H, H-3′, H-5′), 7.30 (s, 1H, H-8), 3.98 (s, 3H, CH O),
.96 (s, 3H, CH O); APCI MS [M + 1] 300.1. Anal. (C16
‚HCl) C, H, N.
log P Determinations. log P values were estimated from
3
O
2
3
4
3
14
H -
3 2
FN O
(
1
yielded 12 (92%): mp 278-280 °C; H NMR (CDCl
3
) δ 8.70 (s,
H, H-2), 8.09 (d, 1H, H-4′, JH,F ) 10.9), 7.62 (s, 1H, Ar-H),
.26 (s, 1H, H-6′), 7.22 (br s, 1H, NH), 7.06 (d, 1H, H-2′, JH,F
8.0 Hz), 7.00 (s, 1H, Ar-H), 4.24 (m, 4H, OCH CH ), 1.55
m, 6H, OCH CH ); HREIMS calcd for C19 m/z (M+)
95.12569, found 395.12596. Anal. (C19 ·HCl) C, H,
the log k′
w
values determined by HPLC chromatography
1
7
44
following the procedure of Minick. The solvents were HPLC-
grade methanol, 1-octanol, n-decylamine, and distilled, deion-
)
2
3
ized water. The standards [p-anisidine (t
R
) 0.95), acetophe-
) 2.26), naphthalene (t
R
) 4.88)] were obtained from Aldrich and
(
2
3
17 4 3 2
H F N O
H F N O
17 4 3 2
none (t
)
R
) 1.58), p-bromoaniline (t
R
R
3
3.30), pyrene (t
N.
used without further purification. A ThermoQuest HPLC
system equipped with an autoinjector, pump, diode-array UV/
vis detector, and an Es Industries MC8 column (4.6 × 150 mm,
4
-[3′-Fluoro-5′-(trifluoromethyl)anilino]-6,7-dimethoxy-
quinazoline Hydrochloride (13). Similar treatment of 9
150 mg, 0.67 mmol) with 3-fluoro-5-(trifluoromethyl)aniline
(
5µ, 60Å) was used for these measurements. The organic mobile
1
yielded 13 (19%): mp 269-270.5 °C; H NMR (CDCl
s, 1H, H-2), 8.08 (d, 1H, H-4′, JH,F ) 10.8), 7.63 (s, 1H, Ar-H),
3
) δ 8.72
phase was methanol containing 0.25% v/v 1-octanol. The
aqueous mobile phase was octanol-saturated water containing
(
7
.38 (br s, 1H, NH), 7.27 (s, 1H, H-6′), 7.06 (d, 1H, H-2′, JH,F
8.4 Hz), 7.02 (s, 1H, Ar-H), 4.03 (s, 3H, OCH ), 4.01 (s, 3H,
); APCI MS [M + 1] 368.1 (100). Anal. (C17
HCl) C, H, N.
0
0
.02 M 3-morpholinopropanesulfonic acid (MOPS) buffer,
)
3
.15% v/v n-decylamine, adjusted to pH 7.4. The flow rate was
OCH
3
13 4 3 2
H F N O ‚
set at 1 mL/min. The quinazolines and standards were
dissolved in methanol to a final concentration of approximately
0
4
-[(4′-Fluorobenzyl)amino]-6,7-diethoxyquinazoline Hy-
.1 mg/mL and 10 µL was injected onto the column. The
drochloride (14). Similar treatment of 8 (66.9 mg, 0.26 mmol)
column void volume was estimated from the retention time of
uracil, which was included as a nonretained internal reference
standard with each injection. The log k′ was determined by
w
linear extrapolation of the compound residence time (retention
volume less void volume) versus the methanol concentration
over a range of 60 to 85% methanol mobile phase.
with 4-fluorobenzylamine yielded 14 (84%): mp 238.5-240 °C;
H NMR (CDCl ) δ 8.56 (s, 1H, H-2), 7.37 (dd, 2H, H-2′,H-6′,
3
1
J
H,H ) 8.2, JH,F ) 5.5), 7.18 (s, 1H, Ar-H), 7.03 (t, 2H, H-3′,
H-5′, JH,F ) JH,F ) 8.6 Hz), 6.84 (s, 1H, Ar-H), 5.53 (br s, 1H,
NH), 4.81 (d, 2H, -CH -, J ) 5.4), 4.20 (q, 2H, OCH CH , J
7.0 Hz), 4.12 (q, 2H, OCH CH , J ) 7.0 Hz), 1.51 (t, 3H,
OCH CH , J ) 7.0 Hz), 1.49 (t, 3H, OCH CH , J ) 7.0 Hz); EI
MS [M + 1] 341 (100). Anal. (C19 ) C, H, N.
-[(4′-Fluorobenzyl)amino]-6,7-dimethoxyquinazo-
line Hydrochloride (15). Similar treatment of 9 (320 mg,
2
2
3
)
2
3
Biological Methods. Cell Lines and Cell Culture. The
CEM human T lymphocyte cell line engineered to express
ErbB4 (CEM/4) and its culture conditions have been described
2
3
2
3
H
3 2
20FN O
5
1,52
4
previously.
Briefly, these cells were propagated in RPMI
supplemented with 10% heat-inactivated fetal bovine serum
and 300 µg/mL G418. The BaF3 mouse lymphoid cell lines
engineered to express either EGFR (BaF3/EGFR) or ErbB2
and ErbB3 together (BaF3/2+3) and the culture conditions for
0
2
.1.43 mmol) with 4-fluorobenzylamine yielded 15 (22%): mp
50-251 °C; H NMR [(CD ) SO] δ 10.37 (br s, 1H, NH), 8.82
3 2
1
(
s, 1H, H-2), 8.00 (s, 1H, Ar-H), 7.45 (dd, 2H, H-2′,H-6′, JH,H
.4, JH,F ) 5.6), 7.22 (s, 1H, Ar-H), 7.19 (t, 2H, H-3′, H-5′, JH,F
H,F ) 8.8 Hz), 4.91 (d, 2H, -CH -, J ) 5.2), 3.95 (s, 3H,
OCH ), 3.94 (s, 3H, OCH ); APCI MS [M + 1] 314.2 (100). Anal.
·HCl) C, H, N.
-(2′-Fluoroanilino)-6,7-diethoxyquinazoline Hydrochlo-
ride (16a). Similar treatment of 8 (0.024 g, 0.095 mmol) with
-fluoroaniline yielded 16a (61%): mp 220.5-222.0 °C; 1
NMR (CD OD) δ 8.62 (s, 1H, H-2), 7.91 (s, 1H, H-5), 7.57 (dd,
)
5
3
8
these cell lines have been described earlier. Briefly, these
cells were propagated in RPMI supplemented with 10% fetal
bovine serum, 300 µg/mL G418, and 10% medium conditioned
by WeHI cells. This conditioned medium serves as a source
for Interleukin 3.
MCF-10A human mammary epithelial cells and MCF-7,
MDA-MB-231, and MDA-MB-453 human mammary tumor cell
lines were obtained from the American Type Culture Collection
(ATCC). These lines were propagated according to ATCC
recommendations.
)
J
2
3
3
17 3 2
(C H16FN O
4
2
H
3
J ) 7.7, 7.5 Hz, 1H, H-5′), 7.44 (m, 1H, H-4′), 7.32 (d, J ) 7.7
Hz, 1H, H-6′), 7.28 (m, 1H, H-3′), 7.20 (s, 1H, H-8), 4.32 (q,
J ) 6.6 Hz, 2H, CH
.55 (t, J ) 6.6 Hz, 3H, CH
3
CH
2
O), 4.29 (q, J ) 6.6 Hz, 2H, CH
3
CH
2
O),
Inhibition of Receptor Tyrosine Phosphorylation. The
assay for inhibition of ErbB family receptor tyrosine phospho-
1
3
CH O), 1.54 (t, J ) 6.6 Hz,
2

7
454 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
VanBrocklin et al.
rylation was adapted from a previously described protocol.52,53
24 h at 37 °C, and a tyrosine kinase inhibitor dissolved in
DMSO was added to each well in a volume of 10 µL. Each
tyrosine kinase inhibitor was assayed at three to five different
concentrations and each concentration was assayed using three
to four wells of cells. Cells treated with 10 µL of DMSO served
as the solvent control. Cells were then incubated for 48 h at
Briefly, 200 mL cultures of CEM/4, BaF3/EGFR, or BaF3/2+3
6
cells were grown to saturation density (∼10 cells/mL) and
were incubated for 24 h at 37 °C in serum-free medium to
reduce basal levels of receptor tyrosine phosphorylation. The
cells were collected by centrifugation and resuspended in
7
3
serum-free medium at a final concentration of ∼10 cells/mL
37 °C. H-Thymidine (1.5 µCi, Amersham Pharmacia Biotech)
(
∼20 mL of cells). Cells were transferred to microcentrifuge
dissolved in a 1.5 µL of an aqueous solution was added to each
well, and the cells were incubated for an additional 2 h at 37
°C. The culture medium was aspirated from the wells, and the
cells were rinsed once with 1 mL of ice-cold phosphate-buffered
saline (PBS) and once with 1 mL of ice-cold 10% trichloroacetic
tubes in 1-mL aliquots and putative kinase inhibitors were
added to the cells. Each tyrosine kinase inhibitor was tested
at three to five different concentrations. The inhibitors were
dissolved in 5 µL DMSO; hence, cells treated with 5 µL DMSO
were used as a solvent control. Cells were incubated in the
presence of inhibitor for 2 h at 37 °C and then incubated on
ice for 20 min. Chilling the cells reduces the amount of ligand-
3
acid (TCA). Incorporated H-thymidine was precipitated by
incubating the cells for at least 30 min at 4 °C in 1 mL of 10%
TCA. Following incubation, the TCA solution was aspirated
5
3
3
induced receptor downregulation.
from each well and the precipitated (incorporated) H-thymi-
dine was solubilized by incubating the cells for 30 min at 95
Ligand was then added to the appropriate samples at a final
concentration of 100 ng/mL, and the samples were mixed and
incubated on ice for 7 min. Recombinant human epidermal
growth factor (EGF; Sigma) was used as the ligand for EGFR,
whereas neuregulin1â (NRG1â; R&D Systems) was used as
the ligand for ErbB3 and ErbB4. Note that because ErbB3
lacks kinase activity, ligand-induced ErbB2 and ErbB3 phos-
phorylation in the BaF3/2+3 cells is the result of ligand-
induced ErbB2-ErbB3 heterodimerization and ErbB2 kinase
°
C in 500 µL of 3% perchloric acid. The perchloric acid extracts
were transferred to scintillation vials containing 10 mL of
3
Cytoscint scintillation cocktail (ICN). The incorporated H-
thymidine was assayed by scintillation counting on a Packard
3
Tricarb scintillation counter. The amount of H-thymidine
incorporation observed in the cells treated with the solvent
control was divided by two to determine the amount of half-
3
maximal H-thymidine incorporation. Dose-response curves
5
3
for each combination of putative tyrosine kinase inhibitor and
activity. Following incubation with ligand, the cells were
collected by centrifugation, the supernatant was removed by
aspiration, and the cells were resuspended in an isotonic lysis
buffer containing 0.5% NP40/Igepal CA-630 (nonionic deter-
gent; Sigma).
3
cell line were then constructed using the H-thymidine incor-
poration data. The dose-response curves and the half-maximal
3
H-thymidine values were used to calculate the concentration
3
of each inhibitor required to inhibit H-thymidine incorporation
by 50% in a given cell line. This value is reported as the DNA
The cells were incubated for 20 min on ice to permit lysis.
The samples were centrifuged for 10 min at 4 °C to collect the
nuclei and cellular debris. The supernatants (cell lysates) were
transferred to fresh tubes. Concanavalin A Sepharose (Amer-
sham/Pharmacia) beads were added to each sample (35 µL of
a 50% v/v slurry) and the samples were incubated at 4 °C for
synthesis IC50 value.
In Vitro EGFR Binding Assay. EGFR tyrosine kinase
receptor binding was determined by a competitive radiometric
1
25
36
assay using [ I]-4-(3′-iodoanilino)-6,7-dimethoxyquinazoline
as the radiotracer (specific activity ) 590 Ci/mmol). Various
-11
-6
concentrations (10 M-10 M) of the quinazoline compounds
30 min. Concanavalin A Sepharose precipitates the cellular
were prepared in binding buffer (10 mM HEPES, 1mM EDTA,
glycoproteins, which include ErbB family receptors. The
precipitated glycoproteins were washed three times with 500
µL of ice-cold lysis buffer and then were eluted by boiling the
beads for 5 min in 80 µL of reducing SDS protein sample
buffer. The beads were collected by centrifugation and half of
the eluted glycoproteins (40 µL) were recovered and resolved
by SDS/PAGE on a 7.5% acrylamide gel.
5
2
mM MgCl , 0.1% BSA, 10 µg/mL leupeptin, 10 µg/mL
pepstatin, 0.5 µg/mL aprotin, and 200 µg/mL bacitracin (pH
.4)). Commercially available (Receptor Biology, Beltsville,
7
MD) A431 human carcinoma cell membrane were diluted in
ice-cold binding buffer (50 µL of 0.06 µg/µL stock solution)and
homogenized with a hand-held homogenizer. This preparation
was added to the buffer solution followed by the addition of 1
µCi of the radiotracer to initiate the binding assay. The
mixture was incubated at room temperature in the dark for
The resolved glycoproteins were electroblotted onto nitrocel-
lulose (BiotraceNT, Gelman Sciences). The resulting blot was
blocked by incubation for 45 min at room temperature in a
solution consisting of 5% bovine serum albumin (Sigma)
dissolved in Tris-buffered normal saline (TBS) supplemented
with 0.05% Tween-20 (TBS-T). The blot was then probed with
a mouse monoclonal antiphosphotyrosine antibody (4G10,
Upstate Biotechnology). The blot was washed with TBS-T five
times for 6 min each, and primary antibody binding was
detected by probing the blot with a goat anti-mouse antibody
conjugated to horseradish perioxidase (HRP; Pierce). The blot
was washed with TBS-T 12 times for 10 min each, after which
HRP activity was visualized by enhanced chemiluminescence
6
0 min. The incubation was terminated by the addition of 5
mL of ice-cold buffer (10 mM HEPES, 1 mM EDTA, 5 mM
MgCl , and 0.1% BSA (pH 7.4)), and the solutions were filtered
2
through polyethylenamine-soaked (0.5% solution, 30 min)
GF/B filter paper (Brandel, Gaithersburg, MD) using a Brandel
cell harvester, followed by two washes (5 mL each) with wash
buffer. The filter paper was dried and counted for 10 min using
a TM Analytic γ well counter. Nonspecific binding was
determined by adding 1 µM 4-(3′-bromoanilino)dimethox-
yquinazoline to the assay. Inhibition constants at 50% of
specific binding (IC50) were derived from specific binding versus
concentration curves. Triplicate assays were performed for
each compound.
(
ECL; Amersham Pharmacia Biotech). The resulting chemi-
lumigrams were digitized using a Linotype-Hell Jade flatbed
scanner and the amount of receptor tyrosine phosphorylation
was quantified using NIH Image software. The amount of
receptor tyrosine phosphorylation in samples from cells treated
with a putative receptor tyrosine kinase inhibitor was com-
pared to a standard curve generated using samples from cells
treated with DMSO solvent control. This enabled us to
determine the concentration of a given tyrosine kinase inhibi-
tor that was necessary to cause a 50% reduction in receptor
tyrosine phosphorylation. This value is reported as the receptor
tyrosine phosphorylation IC50 value.
Acknowledgment. This work was supported in part
by the Director, Office of Science, Office of Biological
and Environmental Research, Medical Science Division
of the U.S. Department of Energy under contract No.
DE-AC03-76SF00098, the Army Medical Research and
Materiel Command, U.S. Department of Defense under
grant no. DAMD17-98-1-8064 (H.F.V.) and grant
no. DAMD17-00-1-0415 (D.J.R.), NIH National Cancer
Institute under grant no. R21CA79823 (H.F.V.),
R01CA094253 (H.F.V.), and R21CA80770 (D.J.R.) and
the California Breast Cancer Research Program under
grant no. 4IB-0059 (H.F.V.). Support for J.K.L. was
Inhibition of Cellular DNA Synthesis. The assay for
inhibition of cellular DNA synthesis was adapted from a
5
4
previously described protocol. Briefly, human mammary
(
tumor) cells were seeded in 1-mL aliquots into 24-well culture
5
dishes at a density of 10 cells/well. Cells were incubated for

EGFR Inhibitors as Imaging Probes
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7455
provided by NIH National Cancer Institute training
grant no. T32CA66527 through the UCSF Department
of Radiology (Randall Hawkins, M.D., Ph.D., P.I.). The
authors wish to thank Dr. Scott Taylor for his helpful
discussion and input on the radiometric binding assay.
The authors also wish to thank Stephen Hanrahan for
the preparation of the iodine-125-labeled quinazolines
for the radiometric binding assay.
(23) Capala, J.; Barth, R. F.; Bailey, M. Q.; Fenstermaker, R. A.;
Marek, M. J. et al. Radiolabeling of epidermal growth factor with
9
9mTc and in vivo localization following intracerebral injection
into normal and glioma-bearing rats. Bioconjugat Chem. 1997,
, 289-295.
24) Rusckowski, M.; Qu, T.; Chang, F.; Hnatowich, D. J. Technetium-
8
(
(
9
9m labeled epidermal growth factor-tumor imaging in mice. J.
Pept. Res. 1997, 50, 393-401.
25) Yang, W.; Barth, R. F.; Leveille, R.; Adams, D. M.; Ciesielski,
M. et al. Evaluation of systemically administered radiolabeled
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Neurooncol. 2001, 55, 19-28.
26) Reilly, R. M.; Kiarash, R.; Cameron, R. G.; Porlier, N.; Sandhu,
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(
Supporting Information Available: Results from el-
emental analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
4
1, 429-438.
(
27) Remy, S.; Reilly, R.; Sheldon, K.; Gariepy, J. A new radioligand
1
11
for the epidermal growth factor receptor:
In labeled human
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