Synthesis and characterization of rhenium and technetium-99m tricarbonyl complexes bearing the 4-[3-bromophenyl]quinazoline moiety as a biomarker for EGFR-TK imaging

Article abstract of DOI:10.1016/j.ejmech.2009.04.033

Aiming at the development of technetium-99m (^99mTc) complexes for early detection and staging of EGFR positive tumors, the tyrosine kinase inhibitor 6-amino-4-[(3-bromophenyl)amino]quinazoline was derivatized with pyridine-2-carboxaldehyde to g

Full text of DOI:10.1016/j.ejmech.2009.04.033

European Journal of Medicinal Chemistry 44 (2009) 4021–4027
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and characterization of rhenium and technetium-99m tricarbonyl
complexes bearing the 4-[3-bromophenyl]quinazoline moiety as a biomarker
for EGFR-TK imaging
,
a
c
Athanasia Bourkoula a, Maria Paravatou-Petsotas a , Apostolos Papadopoulos , Isabel Santos ,
Hans-Jurgen Pietzsch d, Evangelia Livaniou a, Maria Pelecanou b, Minas Papadopoulos a,
Ioannis Pirmettis a
*
a Institute of Radioisotopes–Radiodiagnostic Products, National Centre for Scientific Research ‘‘Demokritos’’, 15310 Athens, Greece
b Institute of Biology, National Centre for Scientific Research ‘‘Demokritos’’, 15310 Athens, Greece
c
´
´
Departamento de Quımica, ITN, Estrada Nacional 10, 2686-953 Sacavem Codex, Portugal
d Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, P.O. Box 510 119, 01314 Dresden, Germany
a r t i c l e i n f o
a b s t r a c t
Aiming at the development of technetium-99m (99mTc) complexes for early detection and staging of EGFR
positive tumors, the tyrosine kinase inhibitor 6-amino-4-[(3-bromophenyl)amino]quinazoline was deriv-
atized with pyridine-2-carboxaldehyde to generate the imine 6-(pyridine-2-methylimine)-4-[(3-bromo-
phenyl)amino]quinazoline suitable for reacting with the fac-[99mTc(CO)3]þ core as an N,N bidentate ligand.
The labelling was performed in high yield (>90%) by ligand exchange reaction using fac-
Article history:
Received 3 March 2009
Received in revised form
13 April 2009
Accepted 18 April 2009
Available online 3 May 2009
[
99mTc(OH2)3(CO)3]þ as precursor. The 99mTc complex was characterized by comparative HPLC analysis
using the analogous rhenium (Re) complex as reference. The Re complex was prepared by ligand
exchange reaction using the fac-[ReBr3(CO)3]2ꢀ as precursor and was fully characterized by NMR and IR
spectroscopies and elemental analysis. In vitro studies indicate that both the ligand and its Re complex
inhibit the EGFR autophosphorylation (IC50: 17 ꢁ 3.7 and 114 ꢁ 23 nM respectively) in intact A431 cells,
Keywords:
Technetium
Rhenium
4-Anilinoquinazoline
Tyrosine kinase inhibitors
Epidermal growth factor receptor
bind the receptor in a reversible mode, and inhibit A431 cell growth (IC50: 5.2 ꢁ 1.1 and 2.0 ꢁ 0.98
mM
respectively). Biodistribution of the 99mTc complex in healthy animals showed a rather fast blood and soft
tissue clearance between 1 and 15 min p.i. with excretion occurring mainly via the hepatobiliary system.
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
transduction pathways. Since tyrosine phosphorylation is a primary
pathway whereby mitogenic signals are transduced through the cell
The epidermal growth factor receptor (EGFR) belongs to the HER
family of transmembrane receptors with intrinsic protein kinase
activity involved in cell proliferation, differentiation, cell migration,
metastasis, angiogenesis and reduced responses to chemotherapy.
These receptors share a similar structure consisting of an extracel-
lular ligand-binding domain, a short hydrophobic transmembrane
region, and an intracellular cytoplasmic domain containing the
locus for tyrosine kinase (TK) activity and regulatory functions.
Interaction of EGFR with its natural ligands, such as the epidermal
growth factor (EGF), leads to dimerization of the ligand–receptor
complexes, followed by autophosphorylation of the receptor at the
intracellular tyrosine kinase domain. The autophosphorylation
serves as a biochemical trigger for several downstream signal
membrane to the nucleus, mutational activation or overexpression
of receptor tyrosine kinases is linked to malignant transformations.
In particular, overexpression of the EGFR and its enhanced signalling
has been associated with the initiation, progression and invasive-
ness of a great spectrum of human cancers. Overexpression of EGFR
occurs in almost 100% of the head and neck tumors, followed by
pancreatic and renal cell carcinomas, colorectal, breast, ovarian,
prostate, bladder, non-small-cell lung cancer, and glioblastomas, etc.
Also high expression of EGFR is frequently observed in many
different types of epithelial cancers [1,2].
Due to its properties, the EGFR emerged in recent years as an
attractive target for the development of anticancer pharmaceuticals
that can specifically bind the receptor and inhibit its TK activity and
downstream signalling [1,3,4]. Monoclonal antibodies (MAbs)
directed against the external domain of the receptor [5,6] and
small-molecule TK inhibitors (TKIs) that bind to the intracellular
* Corresponding author. Tel.: þ30 210 6503837; fax: þ30 210 6524480.
E-mail address: mparavatou@rrp.demokritos.gr (M. Paravatou-Petsotas).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.033

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A. Bourkoula et al. / European Journal of Medicinal Chemistry 44 (2009) 4021–4027
tyrosine kinase region [7–9] are two classes of drugs approved for
clinical use or in preclinical/clinical development for this purpose.
Small-molecule TKIs target the catalytic kinase domain of EGFR and
bind to the ATP-binding pocket of the cytoplasmic part of the
receptor to inhibit intracellular receptor autophosphorylation,
thereby preventing the initiation of signalling cascade. Several
classes of tyrosine inhibitors have been reported [10–12], among
which the 4-anilinoquinazoline derivatives are the most potent and
selective as well as the most investigated so far [13–18].
Furthermore, considerable interest arose in the use of radio-
labelled EGFR inhibitors for molecular imaging of tumors over-
expressing the EGFR receptor and even for tumor radiotherapy.
Several reversible and irreversible inhibitors, particularly from the
4-anilinoquinazoline class, were labelled with positron emitters,
such as fluorine-18, carbon-11 and iodine-124, and their potential
as PET imaging agents was evaluated [19,20].
The yield of the reaction was 70% and all analytical data were
consistent with the expected structure. Compound 4 was synthe-
sized in four steps starting from 4-hydroxyquinazoline, as depicted
in Scheme 1. All compounds were characterized by 1H NMR and
elemental analysis.
2.1.2. Synthesis of the rhenium complex [ReBr(5)(CO)3] (6)
The synthesis of complex 6 (Scheme 2) was effected by ligand
exchange reaction using equimolar quantities of ligand 5 and the
organometallic Re–tricarbonyl precursor [NEt4]2[ReBr3(CO)3]. HPLC
analysis of the reaction mixture showed the formation of a single
product with an Rt of 15.6 min, clearly differentiated from the Rt of
the ligand at 14.2 min. Complex 6 was isolated as an orange powder
in high yield (75%) and was fully characterized by IR and NMR
spectroscopies and elemental analysis.
The same complex was also obtained by using a previously
reported two step method [22,23]. In the first step pyridine-2-
carboxaldehyde reacted with the precursor [NEt4]2[ReBr3(CO)3]
forming the intermediate complex 8 that was not isolated. In the
second step 6-amino-4-[(3-bromophenyl)amino]quinazoline (4)
was added to the reaction mixture. The reaction was monitored by
HPLC where it was shown that after 17 h at room temperature
complexation of 4 was almost quantitative. Complex 6 was isolated
in high yield (75%) as orange crystals.
The almost ideal properties of 99mTc for SPECT imaging and its
widespread availability in comparison to PET isotopes justify the
search for 99mTc-labelled quinazolines. Recently, the synthesis and
in vitro evaluation of Re and 99mTc complexes, in which the 4-ani-
linoquinazoline pharmacophore was coupled to tailor made
chelators suitable for the stabilization of either the MIII (M ¼ Re and
99mTc) core with a ‘‘4 þ1’’ mixed-ligand system or of the tricarbonyl
fac-[MI(CO)3]þcore, were presented [21]. It was shown that the Re
complexes retain to a substantial degree the pharmacological
properties of the 4-anilinoquinazoline pharmacophore, despite the
presence of their organometallic core. These encouraging results
prompted us to continue the investigation for useful 99mTc probes
for early detection and staging of EGFR positive tumors. In this
study we present the synthesis, characterization and biological
evaluation of new Re (6) and 99mTc (7) complexes with the 6-
(pyridine-2-methylimine)-4-[(3-bromophenyl)amino]quinazoline
(5) suitable for acting as a bidentate N,N chelator with the fac-
[M(CO)3]þ (M ¼ Re, 99mTc) core.
The infrared spectrum of complex 6 shows strong bands in the
2023–1903 cmꢀ1 range, attributed to the symmetric and asym-
metric stretch bands of the C^O bond, demonstrating the presence
of the tricarbonyl core in the molecule [21,24–27].
Complex 6 is stable in the solid state as well as in solution for
months, as shown by HPLC and NMR.
2.2. NMR studies
1H and 13C chemical shift assignments for ligand 5 and complex
6 are reported in Table S1. The 1H–13C correlation (HSQC) spectrum
of complex 6 is presented in Fig. 1. Coordination of the ligand
around the metal core results in downfield shifts of 0.7 ppm for the
imine H-6 proton and of 0.3–0.4 ppm for the protons of the pyr-
idinyl moiety, in accordance with coordination of the imine and
pyridinyl nitrogens. Smaller downfield shifts are also noted for the
protons of the quinazolinyl moiety. In the 13C spectrum of complex
6, downfield shifts are noted for the carbons close to the coordi-
nating nitrogens; e.g., the C-6 carbon of the imine moiety is shifted
2. Results
2.1. Chemistry
2.1.1. Synthesis of 6-(pyridine-2-methylimine)-4-[(3-
bromophenyl)amino]quinazoline (5)
Ligand 5 was synthesized by reacting 6-amino-4-[(3-bromo-
phenyl)amino]-quinazoline (4) with pyridine-2-carboxaldehyde.
Br
Cl
OH
N
OH
N
Br
HN
N
O2N
O2N
O2N
HNO3/H2SO
NH2
4
SOCl2
N
N
N
N
N
1
3
2
HN
Br
HN
N
Br
N
CHO
H2N
Fe/CH3COOH
N
N
N
N
N
4
5
Scheme 1.

A. Bourkoula et al. / European Journal of Medicinal Chemistry 44 (2009) 4021–4027
4023
15.8
99mTc- complex 7 (rad)
Re complex 6 (UV)
Br
N
Br
Br
OC
OC
NCHO
OC
OC
O
Re
Re
OC
Br
8
OC
5
15.6
4
3
17
20
4
5
18
19
2
1
16
15
HN
6
N
H2O
Br
14
9
12
OH2
OH2
OC
OC
N
OC
OC
13
7
5
0
5
10
15
minutes
20
25
30
35
99mTc
N
M
X
11
8
10
N
CO
CO
Fig. 2. Reverse phase HPLC chromatograms after injection of: (top) reaction mixture of
labelling with 99mTc at RT for 20 min, gamma trace; (bottom) rhenium complex 6, UV
trace at 254 nm. Re complex 6 Rt ¼ 15.6 min, 99mTc complex 7: Rt ¼ 15.8 min.
6: M = Re, X = Br
7: M = 99mTc, X = Cl
reductant and ‘‘in situ’’ source of CO [28]. The reaction was moni-
tored by HPLC (Rt of pertechnetate 3.0 min, Rt of precursor 5.5 min,
under the employed conditions) and was quantitative (>95%).
Reaction of ligand 5 with the precursor fac-[99mTc(OH2)3(CO)3]þ
at room temperature for 20 min resulted in the formation of the
99mTc complex 7. HPLC analysis showed the formation of a major
product (85–90%) eluting at 15.8 min and a minor complex (w10%)
eluting at 14.8 min (Fig. 2). Corroboration of the structure of the
99mTc complex was effected by comparative HPLC analysis using the
analogous stable Re complex. As shown in Fig. 2 the Re complex
elutes at 15.6 min; therefore the peak at 15.8 min is assigned to the
analogous [99mTcCl(5)(CO)3] complex 7 having a chlorine instead of
bromine in the sixth coordination site.
Scheme 2.
downfield by 8.4 ppm upon coordination. In addition, the three
peaks at 186.7–196.9 ppm are characteristic for the presence of the
tricarbonyl metal core and provide further evidence for the
formation of the complex.
2.3. Preparation of 99mTc complex 7
Labelling of ligand 5 with 99mTc was carried out by ligand
substitution using the fac-[99mTc(OH2)3(CO)3]þ tricarbonyl complex
as precursor. The tricarbonyl precursor was prepared by the addi-
tion of pertechnetate to a commercially available Isolink kit that
contains sodium boranocarbonate, which acts simultaneously as
The stability of the 99mTc complex 7 was monitored by HPLC for
24 h and no significant decomposition was observed (Table 1). Its
stability in the presence of histidine and cysteine at a molar ratio of
1/1000 was also monitored by HPLC. As shown in Table 1, more than
90% of the complex remained intact after 24 h incubation with
either cysteine or histidine.
2.4. Biological evaluation
2.4.1. Inhibition of EGFR phosphorylation and reversibility tests
The compounds 4–6 were evaluated for their potency to inhibit
EGFR phosphorylation in various concentrations by immunoblot-
ting in intact A431 cells that overexpress the EGFR (Fig. 3). The
percentage of EGFR phosphorylation was calculated and dose
response curves were performed. IC50 values of the three
compounds indicating the concentration (nM) needed to inhibit
phosphorylation by 50% are presented in Table 2. In order to
characterize the type of binding of the compounds as reversible or
irreversible, the inhibition of phosphorylation was also determined
Table 1
Percentage of 99mTc complex 7 remaining intact after incubation with 1000 molar
excess of histidine and cysteine at room temperature.
Intact complex (%) after
1 h
4 h
24 h
None
Histidine
Cysteine
99
98
98
98
95
96
96
89
91
Fig. 1. 1H–13C correlation spectrum (HSQC, range dH 9.7–7.1, range 173.3–111.7) of
complex 6 in DMSO-d6 at 25 ꢂC. The numbering of protons is shown in Scheme 2.

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A. Bourkoula et al. / European Journal of Medicinal Chemistry 44 (2009) 4021–4027
Fig. 3. Immunoblotting. The lines A and C represent the inhibition of the phosphorylation of EGFR immediately after removal of the compounds 5 and 6 respectively. The lines B and
D represent the inhibition of the phosphorylation of EGFR 8 h after removal of the compounds 5 and 6 respectively.
8 h after the removal of the compounds and the IC50 values are also
represented in Table 2.
the new ligand 5 by combining a known tyrosine kinase inhibitor,
the 6-amino-4-[(3-bromophenyl)amino]quinazoline (4), with
pyridine carboxaldehyde via the formation of an imine. This ligand
is expected to act as a bidentate chelator of the tricarbonyl fac-
[M(CO)3]þ core with the N of pyridine and the N of imine acting as
donor atoms. Tricarbonyl N,N complexes with other pharmaco-
phore groups have been reported in the literature [23,29].
2.4.2. In vitro inhibition of cell growth
The reference compound 4, the ligand 5, as well as its rhenium
complex 6 were also evaluated for their potency to inhibit the A431
cell growth by the MTT assay. This assay measures the amount of
MTT reduction by mitochondrial dehydrogenase and assumes that
cell viability (corresponding to the reductive activity) is propor-
tional to the production of purple formazan that is measured
spectrophotometrically. Dose–response curves are presented in
Fig. 4 and IC50 values in Table 2.
Spectroscopic and elemental analyses performed on the isolated
Re complex showed that indeed ligand 5 acts as a bidentate
chelator towards the fac-[ReBr3(CO)3]2ꢀ precursor and replaces two
of the three bromine atoms, resulting in the formation of the
desired neutral [ReBr(5)(CO)3] complex 6, as shown in Scheme 2.
The chemistry was subsequently successfully transferred at tracer
level to give the analogous [99mTcCl(5)(CO)3] complex 7 in high
yield and radiochemical purity through the use of the
2.4.3. Biodistribution in mice
The in vivo distribution of the 99mTc complex 7 was evaluated in
healthy mice at 1, 15, 60 and 180 min p.i. and data are summarized
in Table 3. Complex 7 demonstrated a rather fast blood clearance
between 1 and 15 min p.i. followed by a slower clearance between
15 and 180 min p.i. (21.51 ꢁ3.05%, 6.10 ꢁ 0.63% and 2.01 ꢁ0.24% ID/
g, at 1, 15 and 180 min p.i. respectively). Activity in the liver
increases to 19.80 ꢁ 1.55% ID/g, at 15 min and then decreases to
9.53 ꢁ 1.65% ID/g, at 180 min p.i., while the activity in intestines
increases up to 180 min p.i. The activity in the urine (and urinary
bladder) and the stomach remained low during the study
(4.60 ꢁ 0.57%ID and 0.85 ꢁ 0.37% ID/g, respectively, at 180 min p.i.).
[
99mTc(OH2)3(CO)3]þ precursor. In the structure of 7 it is reasoned
that the sixth position is occupied by a chlorine atom – present in
the reaction medium – which replaces one of the H2O ligands of the
precursor. This replacement is a thermodynamically favoured
process, resulting in an overall neutral complex [29,30]. The pres-
ence of chlorine is further supported by the high stability of
complex 7 against histidine and cysteine challenge experiments
(Table 1) given that the two ligands have very high affinity for the
[
99mTc(CO)3]þ core. The alternative presence of a water molecule at
the sixth coordination site, which is easier to substitute due to
electrostatic reasons, would have led in a high exchange with
histidine or cysteine [31].
3. Discussion
Biodistribution of the 99mTc complex in healthy animals (Table 3)
showed a rather fast blood and soft tissue clearance between 1 and
In the search for useful 99mTc probes for early detection and
staging of EGFR positive tumors, we have designed and synthesized
15 min p.i. followed by
a slower clearance between 15 and
180 min p.i. Excretion of the radioactivity occurred almost exclu-
sively through the hepatobiliary system as evidenced by the
decreasing activity in liver and the increasing amount of radioac-
tivity in the intestine. At the same time the activity in the urine (and
urinary bladder) was quite low. Stomach values at 180 min were low
indicating minimal in vivo reoxidation to 99mTcO4ꢀ.
In the design of complexes 6 and 7 the chelating part does not
interfere to a great extent with the biologically active quinazoline
pharmacophore 4 [16] and is expected to present minimal steric
hindrance to the interaction of the complexes with the intracellular
domain of the EGFR. Indeed, the results obtained from the in vitro
biological studies show that both ligand 5 and the rhenium
complex 6 maintain the properties of the parent compound 4.
Specifically, they are able to cross the cell membrane of A431 cells
significantly inhibiting the EGFR autophosphorylation and cell
growth. Comparing ligand 5 to its rhenium complex 6 it is also
apparent that the presence of the metal tricarbonyl core does not
alter significantly the biological activity (IC50 17 nM for 5 vs 114 nM
for 6). The activity of the rhenium complex, 6 is higher than the
Table 2
Inhibition of EGFR phosphorylation and inhibition of A431 cell growth by quina-
zoline derivatives 4–6.
Inhibition of phosphorylation
Immediately after removal
Inhibition of cell
growth IC50
c
8 h after removal
of the inhibitor IC50
a
b
of the inhibitor IC50
4
5
6
3.5 ꢁ 1.1 nM
17 ꢁ 3.7 nM
114 ꢁ 23 nM
>1
>1
>1
mM
mM
mM
4.8 ꢁ 0.2
5.2 ꢁ 1.1
mM
m
M
2.0 ꢁ 0.98
mM
IC50 values are represented as mean ꢁ standard deviation.
a
Concentration needed to inhibit by 50% the autophosphorylation of EGFR in
A431 cells immediately after removal of the compound from the medium (detected
by immunoblotting). Each experiment was performed twice in duplicate.
b
Concentration (>1 mM) needed to inhibit by 50% the autophosphorylation of
EGFR in A431 cells 8 h after removal of the compound from the medium (detected by
immunoblotting). Each experiment was performed twice in duplicate. No exact IC50
values were estimated.
c
Concentration needed to inhibit cell growth by 50%. Each experiment was
performed twice in quadruple.

A. Bourkoula et al. / European Journal of Medicinal Chemistry 44 (2009) 4021–4027
4025
150
A 150
B
125
100
75
50
25
0
125
100
75
50
25
0
-10
-9
-8
-7
-6
-5
Log Concentration (M)
-4
-3
-10
-9
-8
-7
-6
-5
Log Concentration (M)
-4
-3
Fig. 4. MTT assay: dose–effect curves of (A): quinazoline derivative 5, (B): quinazoline derivative 6.
Table 3
ligands and complexes that maintain the activity of the parent
compound and this information may further be exploited in the
design of new EGFR inhibitor 99mTc complexes.
Tissue distribution of radioactivity (%ID/g) after injection of [99mTcCl(5)(CO)3], 7 in
mice.
Tissue
Time
1 min
15 min
60 min
180 min
5. Experimental protocols
Blood
Liver
Heart
Kidneys
Stomach
Intestine
Spleen
Muscle
Lungs
21.51 ꢁ 3.05
8.24 ꢁ 0.60
3.86 ꢁ 0.73
7.29 ꢁ 1.31
0.55 ꢁ 0.03
0.79 ꢁ 0.28
1.53 ꢁ 0.34
0.83 ꢁ 0.14
10.21 ꢁ 3.22
0.40 ꢁ 0.06
0.08 ꢁ 0.03
6.10 ꢁ 0.63
19.80 ꢁ 1.55
1.24 ꢁ 0.21
8.14 ꢁ 0.62
0.85 ꢁ 0.13
4.54 ꢁ 0.57
2.05 ꢁ 0.54
0.44 ꢁ 0.06
3.14 ꢁ 0.53
0.13 ꢁ 0.03
1.57 ꢁ 0.40
4.33 ꢁ 0.79
13.77 ꢁ 0.53
1.21 ꢁ 0.17
7.49 ꢁ 0.56
0.92 ꢁ 0.11
11.90 ꢁ 0.56
1.55 ꢁ 0.22
0.41 ꢁ 0.01
2.38 ꢁ 0.51
0.10 ꢁ 0.02
3.43 ꢁ 0.56
2.01 ꢁ 0.24
9.53 ꢁ 1.65
0.69 ꢁ 0.12
8.16 ꢁ 0.90
0.85 ꢁ 0.37
16.79 ꢁ 1.19
0.82 ꢁ 0.11
0.20 ꢁ 0.04
1.62 ꢁ 0.18
–
5.1. Materials
All laboratory chemicals were reagent grade and were used
without further purification. Solvents for high-performance liquid
chromatography (HPLC) were HPLC-grade. They were filtered
through membrane filters (0.22 mm, Millipore, Milford, MA) and
degassed by a helium flux before and during use.
Brain
Urinea
4.60 ꢁ 0.57
IR spectra were recorded as KBr pellets on a Perkin–Elmer 1600
FT-IR spectrophotometer in the region 4000–500 cmꢀ1. The NMR
spectra were recorded on a Bruker 500 MHz Avance DRX spec-
trometer using (CH3)4Si as the internal reference. Elemental anal-
yses for C, H and N were conducted on a Perkin–Elmer 2400
automatic elemental analyzer. HPLC analysis was performed on
a Waters 600 chromatography system coupled to both a Waters
a
Total excreted urine plus urinary bladder.
previously reported Re–tricarbonyl complexes (Table S2) [21]
indicating an overall improvement in the design of the complexes.
According to the literature [10], quinazoline inhibitors of EGFR
that have a Michael acceptor at the 6-position of quinazoline may
bind irreversibly to the EGFR through formation of a covalent bond
to Cys-773 located in the ATP-binding pocket. Since the presence of
the imine bond at C-6 of the ligand 5 and complex 6 (Scheme 2)
could in principle serve as a site of nucleophilic attack by the Cys
773 thiol, the mode of binding (reversible or not) of the 5 and 6 on
EGFR was further investigated with in vitro experiments. It was
found that phosphorylation of EGFR returns to control level 8 h
after removing the inhibitors 5 and 6, a fact that indicates their
reversible binding (Fig. 3). This is in agreement with the lack of
reactivity of the cysteine thiol against the 99mTc complex 7, as
witnessed in the cysteine challenge experiment. Apparently,
complexes 6 and 7 act as reversible inhibitors since they bind non-
covalently to the catalytic kinase domain of the EGFR.
2487 Dual
l
Absorbance detector and a Gabi gamma detector from
m,
Raytest. Separations were achieved on a Nucleosil C18 (10
m
250 mm ꢃ 4 mm) column eluted with a binary gradient system at
a 1 mL/min flow rate. Mobile phase A was methanol containing 0.1%
trifluoroacetic acid, while mobile phase B was water containing
0.1% trifluoroacetic acid. The elution gradient was 0–1 min 100% B
(0% A), followed by a linear gradient to 90% A (10% B) in 9 min; this
composition was held for another 10 min. After a column wash with
95% A for 5 min, the column was re-equilibrated by applying the
initial conditions (100% B) for 15 min prior to the next injection.
Na99mTcO4 was obtained in physiological saline as
a commercial 99Mo/99mTc generator eluate (Mallinckrodt Medical
B.V., The Netherlands). The carbonyl labelling agent Isolink was
provided by Mallinckrodt Medical B.V., The Netherlands. 6-
Amino-4-[(3-bromophenyl)amino] quinazoline (4) and the
rhenium precursor [NEt4]2[ReBr3(CO)3] have been synthesized
according to literature method [16,32,33]. The radioactive
precursor fac-[99mTc(OH2)3(CO)3]þ was prepared using an Isolink
kit (Mallinckrodt), according to the manufacturers.
The reference compound 4, the ligand 5, as well as its rhenium
complex 6 were also evaluated for their potency to inhibit the A431
cell growth by the MTT assay (Fig. 4 and Table 2). The potency of
ligand 5 (IC50) is similar to that of the parent compound 4 (IC50
5.2 mM and 4.8 mM respectively) while the Re complex 6 is slightly
more potent with an IC50 value of 2.0 mM.
5.2. Synthesis
4. Conclusion
5.2.1. Synthesis of 6-(pyridine-2-methylimine)-4-[(3-
Overall, the biological evaluation of the novel complexes 6 and 7
with the bidentate ligand 5 as EGFR-TK inhibitors is promising and
merits further exploration. This study, in combination with
previous results on 99mTc-labelled quinazolines shows that
attachment of the chelator part at the 6-amino position gives
bromophenyl)amino]-quinazoline (5)
6-Amino-4-[(3-bromophenyl)amino] quinazoline (4) (1.0 g,
1.5 mmol) and pyridine-2-carboxaldehyde (1.5 mL) were trans-
ferred in a Dean–Stark apparatus containing 70 mL benzene. The
mixture was boiled for 2 h and then the benzene was removed. The

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A. Bourkoula et al. / European Journal of Medicinal Chemistry 44 (2009) 4021–4027
yellow residue was washed with hot MeOH and dried under
vacuum. Yield (834 mg, 65%); mp 233–234 ꢂC. NMR assignment in
Table S1. C20H14BrN5 Anal. Calcd: C, 59.42; H, 3.49; N, 17.32. Found:
C, 59.01; H, 3.44; N, 17.16. HPLC, tR: 14.2 min.
5.3.1.1. Inhibition of EGFR phosphorylation in A431 cells and revers-
ibility test protocols. A431 cells were seeded in duplicate in 6/well
plates and grown to 70–80% confluence in supplemented D-MEM
(high glucose). Next day, the cells were incubated in serum-free
medium for 20 h. Afterwards, they were incubated for 2 h with
varying concentrations of inhibitors (1 nM, 10 nM, 100 nM, 200 nM,
5.2.2. Synthesis of rhenium complex [ReBr(5)(CO)3], (6)
Method A. To
a
solution of [NEt4]2[ReBr3(CO)3] (77 mg,
500 nM, 1 mM, 10 mM) and then treated as two different sets: the
0.1 mmol) in 5 mL acetonitrile, 5 (40 mg, 0.1 mmol) was added
under stirring. The solution was refluxed for 10 min, then 2 mL of
distilled water was added and the mixture was refluxed for addi-
tional 30 min. After overnight standing at room temperature the
precipitated dark orange solid was collected and recrystallized with
hot acetonitrile. Yield: 52 mg, 69%. HPLC, tR: 15.6 min.
first set of cells was used to follow the inhibition of EGFR phos-
phorylation (inhibition test), while the second one was used to
investigate whether the inhibition was reversible or irreversible
(reversibility test).
To the first set of cells, EGF (20 ng/mL) was added for 5 min and
then the cells were washed with PBS. Then, 0.2 mL of lysis buffer
(50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM
Method B. To
0.2 mmol) in 5 mL methanol, pyridine-2-carboxaldehyde (21.4 mg,
19 L) (0.2 mmol) was added under stirring. After 1 h a methanolic
a solution of [NEt4]2[ReBr3(CO)3] (154 mg,
PMSF, 50 mg/mL aprotinin, 2 mM sodium orthovanadate, 50 mg/mL
m
leupeptine and 5 mM EDTA) was added in each well and cell
extracts were obtained by scraping cells with a rubber policeman
and heating to 100 ꢂC for 10 min. The second set of cells was
washed with serum-free medium, incubated in D-MEM for 2 h,
washed again with serum-free medium, incubated in D-MEM for
another 2 h, washed with serum-free medium, incubated in D-
MEM for another 4 h, and then stimulated with EGF (20 ng/mL) for
5 min. Afterwards, cell lysates were prepared as described for the
first set of cells.
solution (5 mL) of 6-amino-4-[(3-bromophenyl)amino] quinazo-
line (62.3 mg, 0.2 mmol) was added and the solution was stirred at
room temperature for 17 h. The orange precipitate formed was
collected, washed with water and recrystallized from acetonitrile.
Yield 114 mg, 76%. HPLC, tR: 15.6 min.
Analysis for C23H14Br2N5O3Re: C, 36.62; H, 1.87; N, 9.28; found:
C, 36.30; H, 1.88; N, 8.98. IR (KBr, cmꢀ1): 2023 (s), 1920 (sh, s) and
1903 (br, s). NMR assignment in Table S1.
The total amount of protein in the cell lysates was determined
by the BCA assay [34] in ELISA plates, using a BSA standard curve.
Aliquots of each lysate corresponding to the same protein amount
were loaded onto polyacrylamide gel (8%) and the proteins were
separated by SDS-PAGE electrophoresis and subsequently trans-
ferred to a PVDF membrane. The membrane was immersed in
Ponceau reagent (0.5% Ponceau in 1% acetic acid) for 5 min, washed
with H2O, blocked overnight with TTN containing 5% powdered
milk (1% fat) and incubated 2 h with monoclonal anti-
phosphotyrosine antibody diluted 1/2000 (PY20, Santa Cruz
Biotechnology). The membrane was subsequently washed 3 times
with TTN and incubated with a horseradish peroxidase-conjugated
secondary anti-mouse IgG antibody diluted 1/3000 (Sigma) for 2 h
and finally washed 3 times with TTN. Detection was performed
using a chemiluminescent detection system according to the
manufacturer’s instructions (ECL kit, Amersham). Bands were
quantified using the Band Leader version 3.00 program and the
percentage of EGFR phosphorylation inhibition was calculated.
Both, inhibition and reversibility tests were performed twice for
each of the three compounds 4–6.
5.2.3. 99mTc-labelling
5.2.3.1. Preparation of fac-[99mTc(OH2)3(CO)3]þ precursor. 99mTc
sodium pertechnetate (370–740 MBq, 1 mL) was added to the Iso-
link kit formulation (Mallinckrodt Medical B.V.) containing sodium
tetraborate (2.9 mg), sodium carbonate (7.2 mg), sodium bor-
anocarbonate decahydrate (2.85 mg) and sodium tartrate dihydrate
(8.5 mg) and the mixture was incubated at 95 ꢂC for 20 min. After
cooling, 120
mL 0.1 N HCl was added and complex formation was
checked by HPLC as described in Section 5.1. HPLC, tR: 5.5 min.
5.2.3.2. Synthesis of [99mTcCl(5)(CO)3], (7). A solution (400
the fac-[99mTc(OH2)3(CO)3]þ precursor (pH 10) was added to a vial
containing 400 g of ligand 5, dissolved in 400 L acetonitrile. The
mL) of
m
m
solution was incubated at room temperature for 20 min. The
reaction was monitored by HPLC that showed formation of one
peak at 15.8 min. The identity of the complex was established by
comparative HPLC studies using a sample of the well-characterized
rhenium complex 6 as reference. The stability of the 99mTc complex
at room temperature was studied by HPLC for a period up to 24 h.
5.2.3.3. Challenge with cysteine and histidine. Stability of complex 7
5.3.1.2. Cell proliferation assay. The A431 cells were seeded in 96-
against trans-chelation was checked by cysteine and histidine
well plates at a density of 3000 cells per 100 mL per well and
challenge procedure. Aliquots of 100
added to 900
L of a 10ꢀ2 M histidine and 10ꢀ2 M cysteine solution
in saline, respectively. The samples were incubated at room
temperature for 24 h. At various interval time points, 100 L of each
m
L of [99mTcCl(5)(CO)3] were
incubated for 24 h, so that they were attached to the wells. The
day after seeding, exponentially growing cells were incubated
with various concentrations of the quinazoline derivatives
m
m
(ranging from 1 nM to 100 mM in quadruple) for 72 h. Control
sample was removed and analyzed by HPLC, following the solvent
system used for characterization of the complexes.
consisted of wells with no quinazoline derivatives added. The
medium was removed and the cells were incubated for 4 h in the
presence of 1 mg/mL MTT [35] (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide, Sigma) in RPMI (PAA Laboratories
GmbH) without phenol red for 2 h at 37 ꢂC. The MTT solution was
removed and isopropanol was added to each well to stop the
cleavage of the tetrazolium ring by dehydrogenase enzymes
which convert MTT to an insoluble purple formazan in living
cells. Plates were then agitated at room temperature for about
15–20 min and the level of the coloured formazan derivative was
determined by measuring optical density (OD) on an ELISA reader
(540/620 nm). The results, which were expressed as: mean of the
OD of various replicates ꢃ 100/OD of the control, were plotted
against the corresponding compound concentration in a semi-log
5.3. In vitro biological studies
5.3.1. Cell line
The human epidermoid vulval carcinoma A431 cell line [20],
kindly provided by Dr E. Mishani (Hadassah Hebrew University,
Israel), which overexpresses EGFR was used for the biological
assays. Cells were maintained in high glucose D-MEM medium with
L-glutamine (PAA Laboratories GmbH) and supplemented with 10%
foetal bovine serum (PAA Laboratories GmbH) and penicillin/
streptomycin (PAA Laboratories GmbH) 100 UI/100
CO2 incubator at 37 ꢂC.
mg per mL, in 5%

A. Bourkoula et al. / European Journal of Medicinal Chemistry 44 (2009) 4021–4027
4027
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Appendix. Supplementary data
Detailed 1H and 13C assignments of compounds 5 and 6 in Table
S1. Chemical structures and IC50 values of previously studied Re–
quinazoline complexes [21] in Table S2. Supplementary data asso-
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1016/j.ejmech.2009.04.033.
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