Novel iodine-124 labeled EGFR inhibitors as potential PET agents for molecular imaging in cancer

Article abstract of DOI:10.1016/j.bmc.2004.04.044

The in vivo results with our previously reported irreversible labeled inhibitor [¹¹C]-ML03 suggested that more chemically stable inhibitors, labeled with a longer-lived radioisotope, could be better candidates for molecular imaging of epidermal growth factor receptor (EGFR) positive tumors. On the basis of this hypothesis we synthesized three new irreversible tyrosine kinase (TK) inhibitors with various chemical reactivities. The three new inhibitors were successfully labeled on the anilino moiety with [ ¹²⁴I], starting with the 6-amino-4-[(3-tributylstannylphenyl)amino]- quinazoline (9) precursor. The cell-free results, obtained with these new irreversible inhibitors, indicated that compounds 5 (α-chloro-acetamide derivative) and 6 (4-dimethylamino-but-2-enoic amide derivative) possessed high potencies toward the EGFR with an irreversible inhibition effect. Compound 4 (α-methoxy-acetamide derivative) was found to be less potent, with only a partially irreversible effect. The high potency of compounds 5 and 6 toward the EGFR establishes their potential as PET agents for molecular imaging of EGFR positive tumors. Their prospect as PET biomarkers is further being investigated.

Full text of DOI:10.1016/j.bmc.2004.04.044

Bioorganic & Medicinal Chemistry 12 (2004) 3421–3429
Novel iodine-124 labeled EGFR inhibitors as potential PET agents
for molecular imaging in cancer
Mazal Shaul,a Galith Abourbeh,a,b Orit Jacobson,a Yulia Rozen,a Desideriu Laky,a
Alexander Levitzkib and Eyal Mishania,*
aDepartment of Medical Biophysics and Nuclear Medicine, Hadassah Hebrew University, Hadassah Hospital,
Jerusalem 91120, Israel
bUnit of Cellular Signaling, Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences,
The Hebrew University, Jerusalem 91904, Israel
Received 6 February 2004; accepted 30 April 2004
Abstract—The in vivo results with our previously reported irreversible labeled inhibitor [11C]-ML03 suggested that more chemically
stable inhibitors, labeled with a longer-lived radioisotope, could be better candidates for molecular imaging of epidermal growth
factor receptor (EGFR) positive tumors. On the basis of this hypothesis we synthesized three new irreversible tyrosine kinase (TK)
inhibitors with various chemical reactivities. The three new inhibitors were successfully labeled on the anilino moiety with [124I],
starting with the 6-amino-4-[(3-tributylstannylphenyl)amino]-quinazoline (9) precursor. The cell-free results, obtained with these
new irreversible inhibitors, indicated that compounds 5 (a-chloro-acetamide derivative) and 6 (4-dimethylamino-but-2-enoic amide
derivative) possessed high potencies toward the EGFR with an irreversible inhibition effect. Compound 4 (a-methoxy-acetamide
derivative) was found to be less potent, with only a partially irreversible effect. The high potency of compounds 5 and 6 toward the
EGFR establishes their potential as PET agents for molecular imaging of EGFR positive tumors. Their prospect as PET biomarkers
is further being investigated.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
undergoing clinical Phase 3 trials. Consequently, there
has been a growing interest in the use of EGFR-TK
inhibitors as radiotracers for molecular imaging of
EGFR overexpressing tumors by nuclear medicine
modalities6–9 such as positron emission tomography
(PET). PET is based on the use of short half-life posi-
tron-emitting isotopes, such as 11C (t1=2 20.39 min), 18F
(t1=2 109.8 min), 15O (t1=2 2.037 min), and 13N (t1=2
9.965 min) produced by an in house cyclotrons. Fol-
lowing injection of a suitable biomarker, the PET scan
provides a mapping of the biomarker distribution and
hence of a specific receptor, transporter or enzyme in the
human body.
The epidermal growth factor receptor (EGFR, Erb-B1)
belongs to a family of proteins, involved in the prolif-
eration of normal and malignant cells.1 Overexpression
of the EGFR is a hallmark of many human tumors, such
as breast cancer, glioma, laryngeal cancer, carcinoma of
the head and neck, and prostate cancer.2–5 Thus, the
EGFR is an attractive target for the design and devel-
opment of compounds that can specifically bind the
receptor, and inhibit its tyrosine kinase (TK) activity
and its signal transduction pathway in cancer cells. Such
compounds may serve as therapeutic or diagnostic
agents. The EGFR reversible inhibitor, Iressaꢁ (Fig. 1),
was recently approved by the FDA for treatment of
NSCLC and prostate cancer, and several other anti-
EGFR targeted molecules, such as Tarcevaꢀ (Fig. 1)
and the anti-EGFR antibody Erbituxꢀ, are presently
Our research endeavors have been devoted to the
development of PET bioprobes that would target the
EGFR-TK for molecular imaging purposes.10–12 During
the course of this research several reversible and irre-
versible inhibitors, such as ML01 and ML0313–15 (Fig. 1)
were labeled with fluorine-18 and carbon-11, respec-
tively, and their potential as PET biomarkers was
investigated both in vitro and in vivo. In the case of
reversible inhibitors, such as [18F]-ML01, the high
Keywords: PET; Iodine-124; EGFR-TK; Molecular imaging.
* Corresponding author. Tel.: +972-2-6777931; fax: +972-2-642-1203;
e-mail: mishani@md2.huji.ac.il
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.044

3422
M. Shaul et al. / Bioorg. Med. Chem. 12 (2004) 3421–3429
F
O
HN
N
HN
O
O
N
O
O
Cl
O
O
N
N
N
Tarceva (OSI-774)
F
Iressa (ZD-1839)
18F
Cl
Cl
HN
HN
H
N
Cl
11
Cl
MeO
MeO
C
N
N
O
N
N
18F]-ML01
[
11C]-ML03
[
Figure 1. Chemical structure of reversible and irreversible EGFR inhibitors.
cellular concentration of competitive ATP molecules
brought about the fast washout of the labeled inhibitor
from the receptor’s ATP-binding pocket.13 However, the
rapid washout was abrogated using the irreversible
inhibitor, [11C]-ML03, most probably, due to covalent
binding between the double bond of the acryl-amide
group at the 6-position of the quinazoline ring and Cys-
773 at the receptor’s TK domain. Then again, this
unsaturated, chemically reactive bond led to rapid in
vivo metabolism, low bioavailability and, consequently,
low accumulation of the labeled compound in the tu-
mor, resulting in low tumor/blood uptake ratios.14 In
order to decrease the chemical reactivity of this irre-
versible inhibitor, minimize the metabolic rate and in-
crease the tumor uptake of the labeled biomarkers we
modified the functional group attached at the 6-position
of the quinazoline ring of ML03. The acryl-amide group
of ML03 was replaced with a-methoxy-acetamide,
a-chloro-acetamide and 4-dimethylamino-but-2-enoic
amide. Our previous results with [11C]-ML03 indicated
that despite the low initial tumor/blood uptake ratio, it
moderately increased with time. Based on these findings
we chose to label the new inhibitors with the longer-lived
radioisotope, [124I], at the anilino moiety. This particular
radioisotope is becoming increasingly significant in PET
diagnostic use.16–20 It can be produced by an in house
moderate energy cyclotron however required a special
target chamber. It decays (t1=2 ¼ 4:2 days) simulta-
neously by positron emission (25.6%) and by electron
capture (74.4%). Due to its quantity of short-range
Auger electrons (9.2/decay) it has also been discussed as
a potential therapeutic nuclide.
availability of the compound, resulting in moderate
tumor/blood uptake ratios of the radiotracer.14 These
findings suggested that the development of more chem-
ically stable inhibitors, which would be less reactive, and
therefore less susceptible to metabolic modifications,
may improve the in vivo performance of these com-
pounds, and set them as better candidates for molecular
imaging of EGFR positive tumors. The major drawback
of labeling ML03 with 11C is the short half-life of this
isotope (t1=2 ¼ 20:3 min), limiting the in vivo studies to
rather short time periods, of 1–2 h post injection into the
rats. Hence, this set of new, more chemically stable
inhibitors was designated for labeling with 124I. The
substantially longer half-life of this isotope would enable
a prolonged follow up after injection of the radiotracer.
This assumption relies on the fact that following auto-
phosphorylation of the receptor, it is degraded with a
half-life of 20 h,21 thus allowing sufficient receptor-
inhibitor binding time for imaging. In addition, our
previously reported studies with A431 cells revealed that
the inhibitory effect of ML03 upon EGFR autophos-
phorylation was sustained (98% inhibition, 8 h post re-
moval of the compound from the medium),14 implying
that the compound is covalently bound to the receptor,
thus leaving a broad range of time for imaging.
On the basis of these premises we synthesized three new
EGFR inhibitors, all of which belong to the 6-anilino-
quinazoline family. The three derivatives,11 a-methoxy-
acetamide (4), a-chloro-acetamide (5) and 4-dimethyl-
amino-but-2-enoic amide (6) were labeled with iodine-
124 at the anilino moiety. These groups of inhibitors are
capable of maintaining the irreversible binding nature
since the a carbon (compound 5) and the b carbon
(compound 6) are partially positively charged, and thus
sufficiently reactive to nucleophilic attack by the cysteine
moiety at the receptor’s ATP-binding site.11 The syn-
thesis of the nonlabeled compounds is outlined in Figure
2. Compound 3 was used as a basic starting material for
the preparation of the three different groups of inhibi-
tors. It was reacted with a-methoxy-acetylchloride to
2. Results and discussion
The in vivo studies, performed with our previously
reported irreversible labeled inhibitor, [11C]-ML03,
revealed a high metabolic rate and a low tumor

M. Shaul et al. / Bioorg. Med. Chem. 12 (2004) 3421–3429
3423
Cl
N
H
COOH
NH2
NO2
NH2
N
N
1. Formamide
2. SOCl2 DMF(cat.)
NO2
N
NO2
I
N
I
iPrOH
1
2
H
N
Raney-Ni, N2H4,
H2O/iPrOH/EtOH
NH2
N
I
1.B
r
l
N
C
O
C
2.
C
H
3
M
H 2
2
C
e
2
C
H
NH
O
CH
e
M
CO
l
C
C
l
O
C
2
H
C
l
H
H
C
H
N
N
H
N
N
N
N
N
I
N
OMe
I
O
N
O
6
4
H
H
N
N
Cl
N
I
O
N
5
Figure 2. Synthesis of new irreversible EGFR inhibitors.
yield compound 4 with 65% yield, and with a-chloro-
acetylchloride to yield compound 5 with 54% yield.
Compound 6 was obtained after a two-step synthesis
(Fig. 2). Compound 3 was reacted with bromo/chlo-
rocrotonyl chloride followed by a reaction with di-
methylamine to obtain 6 with a 20% yield. Since the
crotonyl chloride was obtained from the bromocrotonic
acid by a reaction with oxalyl chloride, we obtained a
mixture of inseparable bromo/chlorocrotonyl chloride.22
In order to assess the irreversible effect of the com-
pounds on EGFR autophosphorylation, the inhibitors
were incubated with intact A431 cells for 1 h, as de-
scribed in Section 4, and the degree of EGFR phos-
phorylation was measured either immediately after or
8 h post removal of the inhibitor from the medium. As
previously described,23 if 80% inhibition, or more, is
achieved after 8 h, the compound is considered to be
irreversible, while 20–80% inhibition classifies the com-
pound as ‘partially irreversible’. The results in Table 1
indicate that compounds 5 and 6 possess similar
potencies with regard to their inhibitory effect upon
EGFR phosphorylation. Inhibition of phosphorylation
by these two compounds was retained 8 h post removal
of the inhibitors from the medium, reflecting the irre-
versible effect of these inhibitors, which is most likely,
due to covalent binding at the ATP-binding site. The
ability of compound 5 to bind irreversibly indicates that
a chain of four atoms attached to the 6-position at the
The three nonlabeled compounds, 4–6, were evaluated
in a cell-free system by means of ELISA in order to
determine their EGFR autophosphorylation IC50 val-
ues. The assays were performed with A431 cell lysates,
as described in Section 4, and the IC50 values of the three
compounds are summarized in Table 1. Compounds 5
and 6 appeared to be more potent than the a-meth-
oxyacetamide derivative (4), with compound 6 being as
potent as ML03 with regard to EGFR inhibition.
Table 1. IC50 values in the ELISA screen with A431 cell lysates and IC80 values for inhibition of EGFR autophosphorylation in intact A431 cells
immediately after and 8 h post removal of the compound from the medium
Structure
A431 lysate IC50
app
Intact A431 cells
a
a
IC80 range (immediately after removal of the inhibitor)
IC80 range (8 h post removal of the inhibitor)
ML01
ML03
0.208 nM
0.037 nM
72 50 nM
0.1–20 nM
0.05–5 nM
3–5 nM
5–10 nM
<1 lM
4–10 nM
2–10 nM
––
5–10 nM
>80 lM
10–50 nM
2–10 nM
4
5
6
a IC80 is the inhibitory concentration required to inhibit 80% of EGFR phosphorylation.

3424
M. Shaul et al. / Bioorg. Med. Chem. 12 (2004) 3421–3429
H
Cl
N
H2N
N
N
COOH
1. Formamide
O2N
O2N
O2N
N
Br
N
Br
2. SOCl2 DMF(cat.)
iPrOH
NH2
1
7
H
N
H
N
N
Raney-Ni, N2H4,
H2O/iPrOH/EtOH
H2N
H2N
(SnBu3)2
N
N
Br
SnBu3
Pd(PPh3)4, THF
N
8
9
Figure 3. Synthesis of the starting material for radiolabeling with 124I.
quinazoline moiety is not essential. Structurally, a chain
of three atoms, as in compound 5, is sufficient to achieve
covalent binding at the receptor’s kinase domain. With
compound 4, which is much more chemically stable,
partial irreversible binding was observed.
0.1 M NaOH24 at room temperature to yield the [124I]-6-
amino-4-[(3-iodophenyl)amino]-quinazoline [124I]-3 with
50% radiochemical yield. Reaction of the latter with
bromo/chlorocrotonylchloride at 0 ꢂC was followed by
reaction with dimethylamine, which provided [124I]-6
([124I]-ML06) with 23% yield and a total radiosynthe-
sis time, including HPLC purification, of 90 min. Reac-
Compound 9, the starting material for the radiolabeling,
was prepared by the reaction of 4-chloro-6-nitroqui-
nazoline (1) with 6-bromoaniline to yield compound 7
(Fig. 3). The nitro group at the 6-position of the qui-
nazoline ring was reduced using hydrazine hydrate and
RaNi to yield 6-amino-4-[(3-bromophenyl)amino]-qui-
nazoline (8). In the last step compound 9 was obtained
by the reaction of 8 with (SnBu3)2 in the presence of a
catalytic amount of Pd(PPh3)4 under dry conditions.
For the preparation of the starting material, 9, we chose
to work with the bromoaniline rather than with the
iodoaniline derivative since the nitro reduction step with
the bromo derivative gave better yields than the iodo
derivative (deiodination). In addition, had we chosen to
work with the iodo derivative, the specific activity of the
final labeled compounds might have been lower.
tion of
a-chloroacetylchloride at 0 ꢂC provided the desired
124I]-4([124I]-ML07), and [124I]-5 ([124I]-ML08) with 28%
[
124I]-3 with a-methoxyacetylchloride or
[
and 36% overall radiochemical yields, respectively, and
a total radiosynthesis time of 70 min.
3. Conclusion
In order to achieve a better tumor uptake of the irre-
versible EGFR inhibitors and to afford in vivo analyses
at later time points post injection, we successfully de-
signed and synthesized three new irreversible EGFR
inhibitors with varying chemical reactivities. These
inhibitors were radiolabeled with the longer-lived
radionuclide, iodine-124, on the anilino moiety of the
molecule. The inhibitory potency of compounds 5 (a-
chloroacetamide) and 6 (4-dimethylamino-but-2-enoic
The radiosynthesis route is outlined in Figure 4. Com-
pound 9 was reacted with a solution of [124I] NaI in
HN
[
124I]NaI,
0.1M NaOH
124I
H N
2
HN
N
SnBu
3
N
H N
2
N
N
l
C
O
C
[
124I]-3
H
C
M
H
C
2
9
l
C
e
H
C
.
O
r
B
.
O
C
2
1
H
C
2
N
e2
H
M
2
H
C
C
l
O
C
C
l
H
H
N
N
N
N
N
H
124I
H
O
H
N
N
N
H
N
N
N
N
Cl
N
OMe
124I
124I
[
124I]-6, ML06
O
O
[
124I]-4, ML07
[
124I]-5, ML08
Figure 4. 124I labeling of irreversible EGFR inhibitors.

M. Shaul et al. / Bioorg. Med. Chem. 12 (2004) 3421–3429
3425
amide) toward the EGFR in intact A431 cells was found
to be in the nM range, and their inhibitory effect was
demonstrated to be sustained. These data suggest that a
chain of four atoms, attached at the 6-position of the
quinazoline moiety, is not essential for covalent binding.
Structurally, a chain of three atoms is sufficient to
achieve irreversible binding. With compound 4 (a-
methoxyacetamide), the methoxy group at the a posi-
tion of the acetamide group was found to be insufficient
to serve as a leaving group so as to form a covalent bond
between Cys-773 at the receptor’s kinase domain and
the inhibitor, resulting in a partially irreversible effect.
Altogether, these in vitro data reflect the promising
potential of compounds 5 and 6 as future biomarkers for
molecular imaging of EGFR positive tumors. The fea-
sibility of their use as PET biomarkers is currently
investigated.
a two-necked flask and DMF (100 lL) was added. The
mixture was refluxed for 1 h, and then additional
quantities of SOCl2 (10 mL) and DMF (50 lL) were
added. After a 3 h reflux the thionyl chloride was dis-
tilled out, and the purity of the product, 4-chloro-6-ni-
troquinazoline (1), was determined using a reversed-
phase C18 analytical HPLC column (96–98% purity).
The compound was kept at 0 ꢂC, and used without any
1
further purification for the next step. Mp ¼ 130 ꢂC, H
NMR (DMSO-d6) d 8.78 (1H, d, J ¼ 2 Hz), 8.555 (1H,
dd, J1 ¼ 6:7 Hz, J2 ¼ 2 Hz), 8.432 (1H, s), 7.883 (1H, d,
J ¼ 6:7 Hz). HPLC conditions: C18 analytical column,
40% acetate buffer pH ¼ 3.8/60% acetonitrile,
flow ¼ 1 mL/min; rt ¼ 4.95 min.
4.1.2. 6-Nitro-4-[(3-iodophenyl)amino]-quinazoline (2). 4-
Chloro-6-nitroquinazoline (1) (4 g, 23 mmol) and 3-
iodo-aniline (12.57 g, 57 mmol) were dissolved, and
stirred in i-PrOH (40 mL) at 25 ꢂC for 10 min, yielding a
bright-yellow precipitate. The mixture was then ref-
luxed, stirred for an additional 3 h, and cooled. The
solid was filtered, rinsed with i-PrOH (12 mL), and dried
in a vacuum oven at 80 ꢂC to yield 2 (5.99 g, 78%). MS
4. Materials and methods
4.1. General
1
(m=z) 393.2 (MH)þ; H NMR (DMSO-d6) d 10.56 (1H,
All chemicals were purchased from Sigma–Aldrich,
Fisher Scientific, Merck or J. T. Baker. Chemicals were
used as supplied, excluding THF, which was refluxed
over sodium and benzophenone, and freshly distilled
prior to use. [124I]-NaI was purchased as a 0.02 M
solution from Ritverc GmbH, Russia.
s), 9.664 (1H, d, J ¼ 2:4 Hz), 8.784 (1H, s), 8.578 (1H,
dd, J1 ¼ 11:4 Hz, J2 ¼ 2:1 Hz), 8.270 (1H, br s), 7.955
(2H, m), 7.543 (1H, d, J ¼ 8:1 Hz), 7.228 (1H, t,
J ¼ 7:8 Hz); HPLC conditions: C18 analytical column,
45% acetate buffer pH ¼ 3.8/55% acetonitrile,
flow ¼ 1 mL/min; rt ¼ 17.8 min.
Mass spectrometry was performed in EI mode on a
Thermo Quest––Finnigan Trace MS––mass spectrome-
ter at the Hadassah-Hebrew University Mass Spectros-
copy facility. 1H NMR spectra were obtained on a
Bruker AMX 300 MHz instrument. Elemental analysis
was performed at the Hebrew University Microanalysis
Laboratory. HPLC separations were carried out using a
Varian 9012Q pump, a Varian 9050 variable wavelength
detector operating at 254 nm and a Bioscan Flow-Count
radioactivity detector with a NaI crystal. Analyses of the
labeled and unlabeled compounds were performed on a
reversed-phase system using Waters c-Bondapack C18
analytical column (10 lm, 300 · 3.9 mm) with mobile
phase systems, composed of CH3CN/acetate buffer or
47% CH3CN/53% 0.1 M ammonium formate buffer.
4.1.3. 6-Amino-4-[(3-iodophenyl)amino]-quinazoline (3).
Compound 2 (620 mg, 1.58 mmol) was placed in a
flask, and a solution of H2O–EtOH–IPA, 5:45:50
(107 mL) was added. The mixture was heated to 95 ꢂC,
and an additional 50 mL of solvent was added until
complete dissolution. The mixture was cooled to 65 ꢂC,
and RaNi (1/2 Pasteur pipette) and hydrazine hydrate
(153 lL, 3.16 mmol) were added successively until a
green solution was obtained. The reaction was heated to
80–85 ꢂC, and more RaNi (1/2 Pasteur pipette) and
hydrazine hydrate (38 lL, 0.8 mmol) were added. Reflux
was maintained for 15–20 min. The solution was cooled,
and filtered through a layer of Celite (prepared as slurry
in EtOH). The mixture was evaporated to yield com-
1
pound 3 (180 mg, 31.4%). MS (m=z) 363.0 (MH)þ; H
For the preparation of the labeled [124I]-amine-quinaz-
oline we followed the general procedure of John et al.24
6-Nitroquinazolone was prepared according to the
published procedure.25
NMR (DMSO-d6) d 9.365 (1H, s), 8.347 (1H, s), 8.323
(1H, t, J ¼ 2:4 Hz), 7.918 (1H, dd, J1 ¼ 10 Hz,
J2 ¼ 2:4 Hz), 7.524 (1H, d, J ¼ 11:6 Hz), 7.388 (1H, d,
J ¼ 7:2 Hz), 7.318 (1H, d, J ¼ 2:8 Hz) 7.235 (1H, dd,
J1 ¼ 11:6 Hz, J2 ¼ 2:8 Hz), 7.134 (1H, t, J ¼ 10:4 Hz)
5.595 (2H, br s); HPLC conditions: C18 analytical col-
umn, 55% acetate buffer pH ¼ 3.8/45% acetonitrile,
flow ¼ 1 mL/min; rt ¼ 8.3 min.
A431 human epidermoid vulval carcinoma cells were
grown in Dulbecco’s modified Eagle medium (DMEM)
(Biological Industries, Beit Haemek, Israel) supple-
mented with 10% fetal calf serum and antibiotics (pen-
icillin 105 units/L, streptomycin 100 mg/L) at 37 ꢂC, 5%
CO2.
4.1.4.
N-{4-[(3-Iodophenyl)amino]-quinazoline-6-yl}-2-
methoxyacetamide (4). 6-Amino-4-[(3-iodophenyl)-
amino]-quinazoline (3) (100 mg, 0.276 mmol) was dis-
solved in dry THF (20 mL), and cooled to 0 ꢂC. Trieth-
ylamine (65 lL, 48 mg; 0.469 mmol, 1.7 equiv) was
4.1.1. 4-Chloro-6-nitroquinazoline (1). 6-Nitroquinazo-
lone (2 g, 0.01 mmol) and SOCl2 (20 mL) were placed in

3426
M. Shaul et al. / Bioorg. Med. Chem. 12 (2004) 3421–3429
added, and the mixture was kept at 0 ꢂC for an addi-
tional 15 min. Methoxyacetylchloride (43 lL, 51 mg,
0.469 mmol, 1.7 equiv) was added, and the mixture was
stirred at 0 ꢂC for 30 min. Then the temperature was
raised to room temperature, and a solution of saturated
NaHCO3 (30 mL) was added. THF was evaporated, and
the residue was extracted with ethyl acetate (3 · 30 mL).
The organic layer was dried with Na2SO4, and evapo-
rated. The product was purified on a silica gel flash
chromatography column with MeOH–CH2Cl2, 3:97 as
eluent to yield 4 (102 mg, 64.5%). Mp ¼ 159–163 ꢂC; MS
methylamine (2 M in THF, 10 mL) was added dropwise.
The reaction was heated to 80 ꢂC for 1 h, cooled, and
EtOAc (50 mL) and saturated NaHCO3 (50 mL) were
added. The layers were separated, and the organic layer
was washed with brine, dried with MgSO4, and evapo-
rated. The crude material was purified by silica gel flash
chromatography (MeOH–CH2Cl2 5:95) to yield 6
(180 mg, 58%). Mp ¼ 212–216 ꢂC; MS (m=z) 474 (MH)þ;
1H NMR (DMSO-d6) d 10.4 (1H, s), 9.8 (1H, s), 8.8 (1H,
s), 8.57 (1H, s), 8.32 (1H, s), 7.8 (3H, m), 7.47 (1H, d,
J ¼ 8 Hz), 7.19 (1H, t, J ¼ 8 Hz), 6.85 (1H, dt,
J1 ¼ 15 Hz, J2 ¼ 7:2 Hz), 6.37 (1H, d, J ¼ 15 Hz), 3.1
(2H, d, J ¼ 7:2 Hz), 2.19 (6H, s). Anal. Calcd:
[C20H20IN5OÆ0.5H2O]: C, 49.79; H, 4.35; N, 14.52.
Found: C, 49.51; H, 4.36; N, 13.89. HPLC conditions:
C18 analytical column, 55% acetate buffer pH ¼ 3.8/45%
acetonitrile, flow ¼ 1 mL/min; rt ¼ 7.54 min.
1
(m=z) 435.0 (MH)þ; H NMR (DMSO-d6) d 10.06 (1H,
s), 9.83 (1H, s), 8.697 (1H, d, J ¼ 3 Hz), 8.571 (1H, s),
8.277 (1H, dd, J1 ¼ 5:7 Hz, J2 ¼ 2:7 Hz), 7.991 (1H, dd,
J1 ¼ 16:8 Hz, J2 ¼ 3:3 Hz), 7.909 (1H, dd, J1 ¼ 16:5 Hz,
J2 ¼ 0:9 Hz), 7.781 (1H, d, J ¼ 16:8 Hz), 7.46 (1H, d,
J ¼ 16:5 Hz), 7.183 (1H, t, J ¼ 14:43 Hz), 4.09 (1H, s),
3.43 (3H, s). Anal. Calcd: C, 46.97; H, 3.45; N, 12.89.
Found: C, 46.19; H, 3.65; N, 12.59. HPLC conditions:
C18 analytical column, 55% acetate buffer pH ¼ 3.8/45%
acetonitrile, flow ¼ 1 mL/min; rt ¼ 10.7 min.
4.1.7. 6-Nitro-4-[(3-bromophenyl)amino]-quinazoline (7) .
This compound was prepared in the same manner as 2,
according to Tsou et al.,22 starting from 4-chloro-6-ni-
troquinazoline (1). Mp ¼ 267–270 ꢂC; MS (m=z) 345
(MH)þ; HPLC conditions: C18 column, 55% acetate
buffer pH ¼ 3.8/45% acetonitrile, flow ¼ 1 mL/min;
rt ¼ 7.54 min.
4.1.5.
N-{4-[(3-Iodophenyl)amino]-quinazoline-6-yl}-2-
chloroacetamide (5). Compound 5 was prepared in the
same manner as 4, starting with compound 3 (138 mg,
0.38 mmol) and chloroacetyl chloride (76 lL,
0.94 mmol). The crude product was purified by silica gel
flash chromatography with MeOH–CH2Cl2, 3:97 as
eluent to yield 5 (90 mg, 54%). Mp>300 ꢂC; MS (m=z)
4.1.8. 6-Amino-4-[(3-bromophenyl)amino]-quinazoline (8).
This compound was prepared in the same manner as 3
starting from 6-nitro-4-[(3-bromophenyl) amino]-qui-
nazoline (7) (590 mg, 1.7 mmol) to yield 8 (332 mg,
62%).Mp ¼ 204 ꢂC; MS (m=z) 315 (MH)þ; HPLC con-
ditions: C18 column, 45% acetate buffer pH ¼ 3.8/55%
acetonitrile, flow ¼ 1 mL/min; rt ¼ 6.41 min.
1
439.0 (MH)þ; H NMR (DMSO-d6) d 10.6 (1H, s), 9.9
(1H, s), 8.71 (1H, s), 8.57 (1H, s), 8.25 (1H, m), 7.82 (2H,
m), 7.45 (1H, d, J ¼ 7:8 Hz), 7.19 (2H, m), 4.34 (2H, s).
Anal. Calcd: C, 43.81; H, 2.76; N, 12.77. Found: C,
43.52; H, 3.18; N, 12.21. HPLC conditions: C18 ana-
lytical column, 55% acetate buffer pH ¼ 3.8/45% aceto-
nitrile, flow ¼ 1 mL/min; rt ¼ 13.1 min.
4.1.9.
6-Amino-4-[(3-tributylstannylphenyl)amino]-qui-
nazoline (9). 6-Amino-4-[(3-bromophenyl)-amino]-qui-
nazoline (8) (300 mg, 0.95 mmol) was dissolved in dry
THF (20 mL), and (SnBu3)2 (1.92 mL, 3.78 mmol) was
added, followed by the addition of Pd(PPh3)4 (547.8 mg,
0.474 mmol) in dry THF (0.5 mL). The mixture was ref-
luxed for 16 h, and the solvent was evaporated. The crude
product was purified over an aluminium oxide 90 column
(70–230 mesh) with hexane–dichloromethane 20:80 fol-
lowed by 100% dichloromethane as eluents to yield 9
(85 mg, 20%); MS (m=z) 527 (M+2H)þ; 1H NMR d
(CDCl3): 8.592 (1H, s), 7.75 (1H, d, J ¼ 8:7 Hz), 7.64 (2H,
m), 7.58 (1H, m), 7.47 (3H, m), 1.567 (6H, m), 1.308 (6H,
m), 1.077 (6H, t, J ¼ 5:7 Hz), 0.919 (9H, t, J ¼ 7:2 Hz);
HPLC conditions: Normal-Phase analytical column,
100% acetonitrile, flow ¼ 1.0 mL/min; rt ¼ 13.59 min.
4.1.6. 4-Dimethylamino-but-2-enoic acid {4-[(3-iodophenyl)-
amino]-quinazoline-6-yl}-amide (6). (2E)-4-Bromo/chloro-
N-4-[(3-iodophenyl)amino]-quinazoline-6-yl-2-butenamide
was prepared in the same manner as 5, starting with 3
(382 mg, 1.055 mmol) and Br/Cl-crotonylchloride (221 mg,
1.213 mmol). The crude material was purified by silica gel
flash chromatography (MeOH–CH2Cl2 5:95) to yield
(2E)-4-bromo/chloro-N-{4-[(3-iodophenyl)amino]-quinaz-
oline-6-yl}-2- butenamide (149 mg, ꢀ30%) as an insepa-
rable mixture of the bromide and chloride in a ꢀ1:1 ratio.
MS (m=z) 509 (M)þ, 465 (MH)þ; 1H NMR (DMSO-d6) d
10.6 (1H, s), 10.24 (1H, s), 8.8 (1H, s), 8.58 (1H, s), 8.27
(1H, s), 7.8 (3H, m), 7.47 (1H, d, J ¼ 8 Hz), 7.19 (1H, t,
J ¼ 8 Hz), 6.96 (1H, br dt, J1 ¼ 15 Hz, J2 ¼ 7:2 Hz), 6.47
(1H, d, J ¼ 15 Hz), 4.38 (2H, d, J ¼ 7:2 Hz). Anal. Calcd
(for a 1:1 ratio): C, 43.27; H, 2.82; N, 11.21; Found:
C, 43.60; H, 2.81; N, 11.12; HPLC condition: C18 ana-
lytical column, 55% acetate buffer/45% acetonitrile,
flow ¼ 1.0 mL/min; rt ¼ 21.69 min (first peak), 24.08 min
(second peak).
4.2. Radiochemistry
4.2.1. [124I]-6-Amino-4-[(3-iodophenyl)amino]-quinazoline
([124I]-3). 6-Amino-4-[(3-tributylstannylphenyl)amino]-
quinazoline (9) (4 mg) was placed in a conical vial,
EtOH (1.2 mL) was added, followed by addition of
0.1 M [124I] NaI (1 mL). 0.1 N HCl (1 mL) and Chlor-
amine-T (1 mg/mL) (1 mL) were added, and the vial was
A
solution of (2E)-4-bromo/chloro-N-4-[(3-iodo-
phenyl)amino]-quinazoline-6-yl-2-butenamide (320 mg,
0.662 mmol) in dry THF (70 mL) was stirred, and di-

M. Shaul et al. / Bioorg. Med. Chem. 12 (2004) 3421–3429
3427
sealed. The reaction was stirred at room temperature for
15 min, after which, sodium metabisulfite (200 mg/mL)
(3 mL), a saturated solution of NaHCO3 (6 mL) and
saline solution (6 mL) were added. After the aqueous
solution was vortexed, and loaded onto a C18 Sep-pak,
the latter was rinsed with water (2.5 mL), dried under
nitrogen for 10 min, and the product was eluted with dry
THF (4 mL). The THF was dried with Na2SO4, filtered
through 0.45 lm filter into a v-vial, and used without
any further treatment for the next step. The purity of the
product was analyzed by a reversed-phase C18 analyti-
cal column (10 lm, 300 · 3.9 mm), eluted with 55% ace-
(4 mL) was cooled to 0 ꢂC for 10 min, and methoxyacet-
ylchloride (200 lL) in dry THF (300 lL) was added.
The reaction mixture was stirred for 30–40 min at 0 ꢂC.
A mixture of ACN–H2O (1:1) (200 lL) was added, and
the solution was evaporated under nitrogen, while
cooling in an iced-water bath, to a volume of 400 lL.
The crude product was purified using an HPLC re-
versed-phase C18 preparative column to yield [124I]-4,
with an overall radiochemical yield of 28%, specific
activity of >6 Ci/mmol (our detection limit) and 99%
radiochemical purity (n ¼ 4). HPLC conditions: C18
preparative column, 60% acetate buffer/40% acetonitrile,
flow ¼ 4.0 mL/min; rt ¼ 22.31 min; HPLC conditions:
C18 analytical column, 55% acetate buffer/45% aceto-
nitrile, flow ¼ 1.0 mL/min; rt ¼ 10.78 min.
tate
buffer/45%
acetonitrile,
flow ¼ 1.0 mL/min;
rt ¼ 8.3 min. In order to measure the radiochemical yield
of this step the THF solution, which contained the
reaction product, was evaporated to a volume of 200 lL,
and injected onto a reversed-phase C18 preparative
column. The average radiochemical yield of [124I]-3 was
50% (n ¼ 7). HPLC conditions: C18 preparative col-
umn, eluted with 60% acetate buffer/40% acetonitrile,
flow ¼ 3.0 mL/min; rt ¼ 10.6 min.
4.2.4.
[
124I]-N-{4-[(3-Iodophenyl)amino]-quinazoline-6-
yl}-2-chloroacetamide ([124I]-5). [124I]-5 was obtained in
the same manner as [124I]-4 by the reaction of compound
[
124I]-3 with chloroacetylchloride (200 lL) in dry THF
(300 lL) with an overall radiochemical yield of 36%
specific activity of >6 Ci/mmol (our detection limit) and
99% radiochemical purity (n ¼ 4). HPLC conditions:
C-18 analytical column, 55% acetate buffer/45% aceto-
nitrile, flow ¼ 1.0 mL/min; rt ¼ 13.16 min; HPLC condi-
tions: C18 preparative column, 55% acetate buffer/
45% acetonitrile, flow ¼ 3.0 mL/min; rt ¼ 20.39 min;
HPLC conditions: C18 analytical column, 55% acetate
4.2.2. [124I]-4-Dimethylamino-but-2-enoic acid {4-[(3-iodo-
phenyl)amino]-quinazoline-6-yl}amide ([124I]-6). [124I]-3 in
THF (4 mL) was cooled to 0 ꢂC for 10 min, and a 0.5 mL
solution of Br/Cl-crotonylchloride22 in dry THF
(182 mg/3 mL) was added. The reaction mixture was
stirred for 30–40 min at 0 ꢂC, and used without any
further treatment for the next step. For measurement of
the radiochemical yield of this step the THF solution,
which contained the reaction product, was evaporated
to a volume of 200 lL, and injected onto a reversed-
phase C18 preparative column. [124I]-(2E)-4-bromo/
chloro-N-{4-[(3-iodophenyl)amino]-quinazoline-6-yl}-2-
butenamide was obtained with an average of 58%
radiochemical yield (n ¼ 4). HPLC conditions: C18
analytical column, 55% acetate buffer/45% acetonitrile,
flow ¼ 1.0 mL/min; rt ¼ 21.69 min (first peak), 24.08 min
(second peak). HPLC conditions: C18 preparative col-
umn, 55% acetate buffer/45% acetonitrile, flow ¼ 4.0 mL/
min; rt ¼ 27.17 min (first peak), 31.29 min (second peak).
buffer/45%
13.16 min.
acetonitrile,
flow ¼ 1.0 mL/min;
rt ¼
4.3. Biology
4.3.1. Production of A431 cell lysates as an EGFR source.
A431 cells were grown in 14 cm Petri dishes to about
90% confluence. The dishes were washed twice with cold
phosphate buffered saline (PBS) pH 7.4, and placed on
ice, prior to the addition of 3.25 mL cold, freshly pre-
pared lysis buffer (50 mM N-2-hydroxyethylpiperazine-
N0-2-ethanesulfonic acid (HEPES) buffer, pH 7.4,
150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM
4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride
(AEBSF), 1 lg/mL aprotinin, 300 lg/mL benzamidine,
10 lg/mL leupeptin, 10 lg/mL soy-trypsin inhibitor) for
10 min. The cells were scraped from the plates with a
[
124I]-(2E)-4-Bromo/chloro-N-{4-[(3-iodophenyl)amino]-
quinazoline-6-yl}-2-butenamide in THF (4 mL) was
cooled to 0 ꢂC for 10 min, and 1 mL of 2.0 M dimeth-
ylamine in THF was added. After 30–40 min of stirring
at 0 ꢂC a mixture of ACN–H2O (1:1) (200 lL) was
added, and the solution was evaporated under nitrogen,
while cooling in an iced-water bath, to a volume of
400 lL. The crude product was purified using an HPLC
reversed-phase C18 preparative column to yield [124I]-6
with an average 45% radiochemical yield, specific
activity of >6 Ci/mmol (our detection limit) and 99%
radiochemical purity (n ¼ 4). HPLC conditions: C18
preparative column, 60% acetate buffer/40% acetonitrile,
flow ¼ 3.0 mL/min; rt ¼ 6.95 min. HPLC conditions: C18
analytical column, 55% acetate buffer/45% acetonitrile,
flow ¼ 1.0 mL/min; rt ¼ 7.54 min.
rubber policeman, homogenized with
a
dounce
homogenizer, and centrifuged (Sorvall centrifuge, rotor
5, 10,000 rpm, 10 min, 4 ꢂC). The supernatant, which
contained the EGFR, was collected and frozen at
)70 ꢂC in aliquots.
4.3.2. ELISA screen. EGFR autophosphorylation IC50
values were obtained by means of an ELISA screen. All
the following incubations were performed at 25 ꢂC with
constant shaking. The final volume for each well was
150 lL. After each step, the plate was washed with
200 lL of double distilled water (·4) and 200 lL TBST
buffer (10 mM Tris–HCl pH 7.4 (TBS), 0.2% Tween 20,
170 mM NaCl) (·1).
4.2.3.
[
124I]-N-{4-[(3-Iodophenyl)amino]-quinazoline-6-
124I]-3 in THF
yl}-2-methoxyacetamide ([124I]-4).
[

3428
M. Shaul et al. / Bioorg. Med. Chem. 12 (2004) 3421–3429
A Corning 96-well ELISA plate was coated with
monoclonal anti-EGFR antibody (mAb108) by incu-
bating overnight (4 ꢂC) in a solution of mAb108 diluted
in PBS pH 8.5. Total mAb108 content per well was
0.75 lg. The plate was washed to remove unbound
mAb108, and PBS containing 5% low-fat milk was ad-
ded to block the unbound sites in the plate for 30 min.
An aliquot of A431 cell lysate was thawed, diluted in
PBS pH 7.4, and added to the plate at a final total
protein concentration of 10 lg/well.
phenol blue, to the cells, scraping them with a rubber
policeman and heating to 100 ꢂC for 10 min. The other
set of cells was incubated for 2 h, washed with PBS,
incubated for another 2 h, washed again, incubated a
further 4 h, and then stimulated with EGF. Cell lysates
were prepared similar to the first set of cells.
4.3.4. Western blot analysis. Protein lysates (30 lg) were
separated by SDS-PAGE (8% polyacrylamide), and
electrophoretically transferred to a nitrocellulose mem-
brane. The membrane was blocked in blocking TBST
for 30 min, and incubated overnight with PY20 anti
phosphotyrosine antibody (Santa Cruz Biotechnology
Inc., Santa Cruz, USA) diluted 1:2000 in blocking
TBST. The membrane was washed thoroughly with
TBST, and incubated for 1 h with a horseradish perox-
idase-conjugated anti-mouse IgG (Jackson Immuno
Research, 1:10,000 dilution in blocking TBST). Finally,
the membrane was washed in TBST (4 · 5 min), and
immunoreactive proteins were visualized using an
enhanced chemiluminescence (ECL) detection reagent.
Quantification of each band was performed using
Adobe Photoshop 5.0 ME and NIH image 1.61/ppc
programs.
After 30 min each inhibitor was added at a range of
concentrations, and for each series, one well was left
with no inhibitor, as a zero inhibition control. All
inhibitors were diluted in TBS/dimethylsulfoxide
(DMSO) to a final DMSO concentration of 0.5% in each
well. After an additional 25 min, and without washing
the plate, ATP–MnCl2 solution was added to each well
to start the reaction, the final concentration being 5 lM
ATP, 5 mM MnCl2. The reaction was allowed to pro-
ceed for exactly 5 min.
To stop the phosphorylation reaction an ethylenedi-
amine tetra-acetic acid (EDTA) solution (pH 8.0, 100mM
final concentration) was added to each well, and after
10 min the plate was washed. Polyclonal antiphosp-
hotyrosine serum (Sugen Inc.) diluted 1:2000 in TBST
containing 5% low-fat milk (blocking TBST) was added,
and the plate was incubated for 45 min.
Acknowledgements
This research was supported by TK Signal, Ltd, Naza-
reth, Israel, and Rotem Industries, Ltd Beer Sheva,
Israel.
For the colorimetric detection of phosphotyrosine con-
tent in EGFR commercial TAGO anti-rabbit peroxi-
dase-conjugated antibody diluted 1:10,000 in blocking
TBST was added to the wells, and allowed to react for
45 min. After washing with TBST only the colorimetric
reaction was initiated by adding 100 lL/well ABTS–
H2O2 in citrate–phosphate buffer pH 4.0 (7.5 mg
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