Rhenium and technetium complexes bearing quinazoline derivatives: Progress towards a 99mTc biomarker for EGFR-TK imaging

Article abstract of DOI:10.1039/b802021c

The quinazoline derivatives (3-chloro-4-fluorophenyl)quinazoline-4,6- diamine (2) and (3-bromophenyl)quinazoline-4,6-diamine (3) were labelled with ^99mTc using the "4 + 1" mixed-ligand system [Tc(NS ₃)(CN-R)] and the tricarbonyl moiety fac-[Tc(CO)₃] ⁺. In the "4 + 1" approach the technetium(iii) is stabilized by a monodentate isocyanide bearing a quinazoline fragment (L ¹, L²) and by the tetradentate tripodal ligand tris(2-mercaptoethyl)-amine (NS₃). In the "4 + 1" approach, ^99mTc-labelling was performed in a two-step procedure, the complexes [Tc(NS₃)(L¹)] (7a) and [Tc(NS₃)(L ²)] (8a) being obtained in about 50-70% yield. In the tricarbonyl approach, the fac-[Tc(CO)₃]⁺ unit is anchored by two different monoanionic chelators bearing the quinazoline derivatives (3-chloro-4-fluorophenyl)quinazoline-4,6-diamine (2) and (3-bromophenyl) quinazoline-4,6-diamine (3). Both chelators have a N₂O donor atom set, but one contains a pyrazolyl ring (L⁵H) and the other contains a pyridine unit (L⁶H). In both cases the conjugation of the quinazoline to the chelator was done through the secondary amine of the potentially tridentate and monoanionic chelators, the corresponding ^99mTc-complexes (10a, 11a) being obtained in quantitative yield. The identities of the ^99mTc-labelled quinazolines (7a, 8a, 10a, 11a) were confirmed by comparison with the HPLC profiles of the analogous Re compounds (7, 8, 10, 11). All these Re complexes were characterized by NMR and IR spectroscopy, elemental analysis and in some cases by MS and X-ray diffraction analysis. In vitro studies indicate that the quinazoline fragments, after conjugation to the cyano group (L¹, L²) or to the pyrazolyl containing chelator (L⁵H), as well as the corresponding Re complexes (7, 8, 10) inhibit significantly the EGFR autophosphorylation and also inhibit A431 cell growth. These two effects were also found for the pyridine-containing chelator (L⁶H) and corresponding Re complex (11), although to a lesser extent. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b802021c

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PAPER
www.rsc.org/dalton | Dalton Transactions
Rhenium and technetium complexes bearing quinazoline derivatives:
progress towards a 99mTc biomarker for EGFR-TK imaging†
Ce´lia Fernandes,a Isabel C. Santos,a I. Santos,*a Hans-Jurgen Pietzsch,b Jens-Uwe Kunstler,b Werner Kraus,c
Ana Rey,d Nikos Margaritis,e Athanasia Bourkoula,e Aris Chiotellis,e Maria Paravatou-Petsotase and
Ioannis Pirmettise
Received 4th February 2008, Accepted 1st April 2008
First published as an Advance Article on the web 13th May 2008
DOI: 10.1039/b802021c
The quinazoline derivatives (3-chloro-4-fluorophenyl)quinazoline-4,6-diamine (2) and
(3-bromophenyl)quinazoline-4,6-diamine (3) were labelled with 99mTc using the “4 + 1” mixed-ligand
system [Tc(NS3)(CN-R)] and the tricarbonyl moiety fac-[Tc(CO)3]+. In the “4 + 1” approach the
technetium(III) is stabilized by a monodentate isocyanide bearing a quinazoline fragment (L1, L2) and
by the tetradentate tripodal ligand tris(2-mercaptoethyl)-amine (NS3). In the “4 + 1” approach,
99mTc-labelling was performed in a two-step procedure, the complexes [Tc(NS3)(L1)] (7a) and
[Tc(NS3)(L2)] (8a) being obtained in about 50–70% yield. In the tricarbonyl approach, the
fac-[Tc(CO)3]+ unit is anchored by two different monoanionic chelators bearing the
quinazoline derivatives (3-chloro-4-fluorophenyl)quinazoline-4,6-diamine (2) and
(3-bromophenyl)quinazoline-4,6-diamine (3). Both chelators have a N2O donor atom set, but one
contains a pyrazolyl ring (L5H) and the other contains a pyridine unit (L6H). In both cases the
conjugation of the quinazoline to the chelator was done through the secondary amine of the potentially
tridentate and monoanionic chelators, the corresponding 99mTc-complexes (10a, 11a) being obtained in
quantitative yield. The identities of the 99mTc-labelled quinazolines (7a, 8a, 10a, 11a) were confirmed by
comparison with the HPLC profiles of the analogous Re compounds (7, 8, 10, 11). All these Re
complexes were characterized by NMR and IR spectroscopy, elemental analysis and in some cases by
MS and X-ray diffraction analysis. In vitro studies indicate that the quinazoline fragments, after
conjugation to the cyano group (L1, L2) or to the pyrazolyl containing chelator (L5H), as well as the
corresponding Re complexes (7, 8, 10) inhibit significantly the EGFR autophosphorylation and also
inhibit A431 cell growth. These two effects were also found for the pyridine-containing chelator (L6H)
and corresponding Re complex (11), although to a lesser extent.
transforming growth factor a (TGFa). After ligand binding, the
Introduction
receptor undergoes dimerisation, forming either homodimers or
The human epidermal growth factor (HER) family consists
heterodimers, followed by internalisation of the receptor–ligand
of four distinct but closely related transmembrane receptors,
complex, which induces tyrosine kinase catalytic activity leading
namely EGFR or HER1, HER2, HER3 and HER4. From all
to autophosphorylation in several tyrosines within the receptor’s
carboxyl-terminal tail.4 These events will trigger a signalling
network that results for example in tumour cell proliferation,
angiogenesis and protection from apoptosis.
these receptors it is known that EGFR is over-expressed and/or
dysregulated in several solid tumors. These receptors normally
possess an extracellular ligand binding domain, a transmembrane
region and an intracellular cytoplasmatic domain with tyrosine
kinase activity.1–3 The most important endogenous stimulatory
ligands for EGFRs are epidermal growth factor (EGF) and
A better understanding of EGFR and of its ligands has
motivated research on new therapeutic strategies for targeting
EGFR and has been reviewed in several papers.2,4–9 The most
studied compounds have been monoclonal antibodies or tyrosine
kinase inhibitors (EGFR-TKI). Monoclonal antibodies act in the
extracellular domain of the receptor and tyrosine kinase inhibitors
bind to the intracellular tyrosine kinase region, competing with
ATP (adenosine triphosphate). Several classes of tyrosine kinase
inhibitors have been reported and, among them, the most potent,
selective and approved are iressa/gefitinib and tarceva/erlotinib
(Chart 1).
aDepartamento de Qu´ımica, ITN, Estrada Nacional 10, 2686-953, Sacave´m
Codex, Portugal. E-mail: isantos@itn.pt
bInstitute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf,
P.O.B. 510 119, 01314, Dresden, Germany
cFederal Institute for Materials Research and Testing (BAM), 12205, Berlin,
Germany
dRadiochemistry Department, Faculty of Chemistry, Universidad de la
Republica, Montevideo, Uruguay
eInstitute of Radioisotopes - Radiodiagnostic Products, National Centre for
Scientific Research “Demokritos”, 15310, Athens, Greece
Tumour imaging of EGFR receptors has been attempted with
monoclonal antibodies or with EGF analogues labelled with99mTc,
111In or 131I.9 Recently, there has also been growing interest
in the use of radioprobes based on tyrosine kinase inhibitors.
† CCDC reference numbers 676393 (for 8) and 676394 (for 9). For
crystallographic data in CIF or other electronic format see DOI:
10.1039/b802021c
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509, Berthold). Ligands and the Re complexes were detected by
UV detection at 254 nm. Separations were achieved on a reversed
phase (RP) Hypersil ODS column (4.0 × 250 mm, 10 lm) eluted
with a binary gradient system with a flow rate of 1.0 mL min−1.
Mobile phase A: aqueous 0.5% TFA solution, mobile phase B:
0.1% TFA solution in acetonitrile; method: t = 0–3 min, 0% B;
3–3.1 min, 0–25% B; 3.1–9 min, 25% B; 9–9.1 min, 25–34% B; 9.1–
20 min, 34–100% B; 20–25 min, 100% B; 25–25.1 min, 100–0% B;
25.1–30 min, 0% B. The 99mTc complexes were also analysed by
TLC using a mixture of methanol (95%)–HCl 6 N (5%).
The compounds N-(2-bromoethyl)-3,5-dimethylpyrazole,24
picolinamine-N,N-diacetic acid (PADA),25 N,N-bis[2-oxo-3-
oxazolidinyl]phosphorodiamidic chloride (BOP-Cl),26 6-amino-4-
[(3-chloro-4-fluorophenyl)amino]quinazoline (2),27 6-amino-4-[(3-
bromophenyl)amino]quinazoline (3),28 the tetradentate tripodal
ligand tris(2-mercaptoethyl)amine (NS3) and the rhenium starting
complexes [ReNS3(PMe2Ph)] and [NEt4]2[Re(Br)3(CO)3] were
prepared as reported elsewhere.29,30 Tris(2-mercaptoethyl)amine
was isolated and applied as the oxalate. The radioactive precursor
fac-[99mTc(CO)3(H2O)3]+ was prepared using an IsoLink kit
(Malinckrodt), according to the manufacturers.
Chart 1
These radioprobes would be very helpful in the selection of
patients for TKI therapy and to monitor their response to
personalized cancer management.4–9 Particularly, those from the
4-anilinoquinazoline class were labelled with short-lived positron
emitters, like 11C,10–17 18F18–21 and 124I,22–23 but up to now, to the
best of our knowledge, no labelling has been attempted with
99mTc.9 However, the widespread availability of single-photon-
emission tomography (SPECT) facilities in comparison with PET,
the worldwide availability of 99Mo–99mTc generators and the almost
ideal properties of 99mTc for SPECT imaging still justify the
interest in 99mTc biocomplexes specific for EGFR-TK, as an
alternative to PET radioligands. In this manuscript, we describe
our attempts to label two quinazoline derivatives with the metal
fragments [M(NS3)(CNR)] and fac-[M(CO)3]+ (M = Re, 99mTc).
One of the quinazoline derivatives is related to PD 153035 (4-[(3-
bromophenyl)amino]-6,7-dimethyoxyquinazoline) and the other
to the recently approved gefitinib.9 We will present the synthesis
and characterization of the quinazoline analogues, their conjuga-
tion to tailor-made ligands for the two metal cores explored and
the synthesis and characterization of the corresponding Re and Tc
complexes. The inhibition of autophosphorylation and cell growth
by the compounds described in this work is also reported.
Synthesis and characterization of N-{4-[(3-chloro-4-
fluorophenyl)amino]quinazolin-6-yl}-3-isocyanopropanamide (L1)
and N-{4-[(3-bromophenyl)amino]quinazolin-6-yl}-3-
isocyanopropanamide (L2)
1. Synthesis of N-formyl b-alanin (1). b-Alanin (10 g,
112 mmol) was dissolved in a mixture of 80 mL of formic acid and
50 mL of acetic anhydride and heated under reflux for 6 h. After
removal of the solvent, the remaining white powder was washed
with diethyl ether and recrystallized from 2-propanol, yielding a
white powder: yield. 45%. Elem. Anal. (C4H7NO3 MW: 117.10)
Calc. C, 41.03%, H, 6.03%, N, 11.96%; Found C, 41.23%, H,
5.86%, N, 12.12%.
Experimental
Unless otherwise stated, all chemicals were of reagent grade and
were used without further purification. NMR (1H, 13C and 19F)
spectra were recorded on Varian Unity 300, 400 or 500 MHz
spectrometers. The NMR spectra were run at room temperature.
1H and 13C chemical shifts are reported relative to residual solvent
signals or TMS as reference. Mass spectrometric measurements
were performed with a Micromass Tandem Quadropole Mass
Spectrometer (Quadro LC) operating in the MS mode. Mass
spectral data were recorded in the positive ESI mode. About
10−4 mol of the sample dissolved in 1.0 mL of methanol or
acetonitrile were injected at a flow rate of 5 mL min−1. Elemental
analyses were performed on a LECO Elemental Analyzer CHNS-
932 or on a EA 1110 CE Instrument automatic analyser. IR spectra
were recorded on a FTIR spectrometer IR 2000 (Perkin-Elmer) or
on a Bruker Tensor 27 spectrometer, using KBr pellets.
2. General procedure for preparation of N-formylquinazolines
4 and 5. 80 mg (640 lmol) of diisopropylethyl amine and
205 mg (640 lmol) of O-(benzotriazol-1-yl)-N,N,Nꢀ,Nꢀ-tetra-
methyluronium tetrafluoroborate (TBTU) were added to 78 mg
(670 lmol) of 1, dissolved in 3 mL of N-methyl-2-pyrrolidone.
After stirring at room temperature for 5 min, 536 lmol of 2 or 3,
dissolved in 4 mL of N-methyl-2-pyrrolidone, was added in one
step. The yellow reaction mixture was stirred at room temperature
for 24 h. After removal of the solvent the product was purified
by column chromatography on silica gel 60 using a mixture of
chloroform–methanol–25% ammonia (80 : 60 : 0.2).
N-{4-[(3-Chloro-4-fluorophenyl)amino]quinazolin-6-yl}-N3-
formyl alaninamide (4). Pale tan solid, yield 30%. MS (ES+):
388.17 [M + H]+. Elem. Anal. (C18H15ClFN5O2 MW: 387.79) Calc.
C, 55.75%, H, 3.90%, N, 18.06%; Found C, 55.46%, H, 3.78%, N,
18.27%.
Chemical reactions were monitored by thin-layer chromatog-
raphy (TLC) on Merck plates precoated with silica gel 60 F254
.
Column chromatography was performed in silica gel 60 (Merck).
Sodium pertechnetate, Na[99mTcO4], was eluted from a commercial
99Mo/99mTc generator (MDS Nordion S.A., Belgium or Mallinck-
rodt), using 0.9% saline solution. High performance liquid
chromatography (HPLC) analysis of the ligands, Re and 99mTc
complexes was performed on a Perkin Elmer system equipped with
a biocompatible quaternary pump (LC 200), a tunable UV/Vis
detector (LC 290, Perkin Elmer) and a radiometric detector (LB
N-{4-[(3-bromophenyl)amino]quinazolin-6-yl}-N3-formyl alan-
inamide (5). Pale tan solid, yield 35%. MS (ES+): 415.20 [M +
H]+. Elem. Anal. (C18H16BrN5O2 MW: 414.26) Calc. C, 52.19%,
H, 3.89%, N, 16.91%; Found C, 52.43%, H, 3.96%, N, 16.77%.
3. General procedure for preparation of isocyanides L1 and L2.
200 lmol of 4 or 5 was dissolved in 10 mL of anhydrous pyridine,
and 70 mg (360 lmol) of p-toluenesulfonyl chloride was added.
3216 | Dalton Trans., 2008, 3215–3225
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−1
=
After being stirred for 6 h at room temperature, the mixture was
transferred into a flask filled with ice. After the ice was slowly
melted, the precipitate was filtered off and washed with water
until no pyridine smell was noticed. The product was purified
by column chromatography on silica gel 60 using a mixture of
chloroform–methanol–25% ammonia (80 : 60 : 0.2) as eluent.
14.9 (CH3), 12.6 (CH3). IR (KBr, cm ): 1601.8 (m(C O)). Elem.
Anal. (C9H14O2N3Na MW: 219.22) Calc: C, 49.31%, H, 6.44%, N,
19.17%; Found: C, 49.36%, H, 8.08%, N, 18.51%.
L4H. Yield 92 mg, 0.29 mmol, 16%: 1H NMR [d, ppm, D2O]:
5.87 (s, 2H, H(4)pz), 4.08 (t, 4H, CH2), 3.34 (s, 2H, CH2CO), 3.18
(t, 4H, CH2), 2.14 (s, 6H, CH3), 2.06 (s, 6H, CH3). 13C NMR [d,
ppm, D2O]: 174.8 (COO), 151.5 (C(3)pz), 144.1 (C(5)pz), 108.0
(C(4)pz), 58.9 (CH2), 55.9 (CH2), 45.6 (CH2), 14.3 (CH3), 12.1
(CH3). Elem. Anal. (C16H25O2N5 MW: 319.41) Calc: C, 60.17%,
H, 7.89%, N, 21.93%; Found: C, 60.33%, H, 8.05%, N, 21.54%.
1
−1
≡
L . Beige solid, yield: 33%. IR (KBr, cm ): 2157 m(–N C).
1H NMR (400 MHz, DMSO-d6): d (ppm) 2.8 (2H, CH2), 3.8
(2H, CN-CH2), 7.4–8.7 (7Harom), 9.9 (NH), 10.4 (NH-C(O)). Elem.
Anal. (C18H13ClFN5O MW: 369.78) Calc. C, 58.47%, H, 3.54%,
N, 18.94%; Found C, 58.61%, H, 3.86%, N, 18.77%.
2
−1
≡
L . Beige solid, yield: 37%. IR (KBr, cm ): 2155 m(–N C).
1H NMR (400 MHz, DMSO-d6): d (ppm) 2.8 (2H, CH2), 3.8
(2H, CN-CH2), 7.3–8.7 (8Harom), 9.9 (NH), 10.5 (NH-C(O)). Elem.
Anal. (C18H14BrN5O MW: 396.24) Calc. C, 54.56%, H, 3.56%, N,
17.67%; Found C, 54.91%, H, 3.84%, N, 17.91%.
Synthesis and characterization of L5H
50 mg (0.25 mmol) of L3H in 2 mL H2O reacted with 100 mg
(0.24 mmol) of 6 in THF (3 mL). The pH was adjusted to 8–
10 by adding NaOH 5 M. The mixture was refluxed and the
pH controlled and adjusted to be maintained between 8 and 10.
The reaction mixture was vacuum dried and the solid purified by
silica gel column chromatography, using as solvent MeOH–25%
NH4OH–CHCl3 and a gradient from 10 : 2 : 88 to 98 : 2 : 0. Yield
45mg, 0.08 mmol, 34%, 1H NMR [d, ppm, CD3OD]: 8.70 (d, 1H,
H(5)), 8.56 (s, 1H, H(2)), 8.00 (d, 1H, H(7)), 7.82 (dd, 1H H(2ꢀ)),
7.67–7.74 (m, 2H, H(8), H(6ꢀ)), 7.28 (t, 1H, (H5ꢀ)), 5.69 (s, 1H,
H(4)pz), 4.24 (t, 2H, CH2), 3.72 (s, 2H, CH2COOH), 3.31–3.39
(m, 4H, 2CH2), 2.78 (t, 2H, CH2) 2.23 (s 3H, Me), 2.06 (s, 3H,
Me). 13C NMR [d, ppm, CD3OD]: 177.3 (CONH), 172.9 (COO),
159.5, 156.1 (d, JF–H 240 Hz), 154.3 (C(3), pz), 148.8, 147.0 (C(5),
pz), 141.7, 138.9, 137.3, 129.3, 128.1, 126.01, 124.2 (d), 121.3 (d,
N-{4-[(3-Chloro-4-fluorophenyl)amino]quinazoline-6-yl}-3-bromo-
propionamide (6). 3-Bromopropanoyl chloride (446 mg,
2.6 mmol) was added slowly to a solution of 6-amino-4-[(3-
chloro-4-fluorophenyl)amino]quinazoline (2) (342 mg, 1.2 mmol)
in dry acetone (15 mL). A yellow precipitate formed and the
reaction mixture was left under stirring for 2 h. The precipitate
was separated by filtration and dried under reduced pressure
yielding 6 as a yellow solid. Yield: (394 mg, 79%). 1H NMR
((CD3)2SO) d (ppm): 3.09 (t, 2H, -CH2-CH2-Br); 3.78 (t, 2H,
-CH2-CH2-Br); 7.55 (t, 1H, H-5ꢀ, J = 8.7Hz); 7.65 (m, 3H, H-6ꢀ);
7.91 (d, 1H, H-8); 7.96 (m, 1H + 1H, H-2ꢀ, H-7); 8.8 (s, 1H, H-2);
1
9.02 (s, 1H, H-5); 10.76 (s, 1H, NH), 11.46 (s,1H,CONH). H
J
F–C 18.5 Hz), 117.4 (d, JF–C 22.2 Hz), 116.6, 112.6, 106.0 (C(4), pz),
NMR (CD3OD) d (ppm): 2.96 (t, 2H, -CH2-CH2-Br); 3.93 (t,
2H, -CH2-CH2-Br); 7.37 (t, 1H, arom); 7.71–7.66 (m, 3H, arom);
8.04–7.84 (m, 1H, arom); 8.76 (s, 1H, arom); 9.01 (s, 1H, arom).
13C NMR (CD3OD) d (ppm): 171.5 (CONH), 161.7, 157.9 (d,
59.5 (CH2), 54.4 (CH2), 51.7 (CH2), 46.7 (CH2), 35.2 (CH2), 13.3
(CH3-pz), 11.1 (CH3-pz). RP-HPLC (UV/Vis detection, 254 nm)
retention time: 16.0 min.
J
F–C 247 Hz), 150.8, 140.7, 136.3, 134.7, 130.7, 128.4, 126.4, 126.5,
Synthesis and characterization of ({[(N-(3-
bromophenyl)amino)quinazolin-6-ylcarbamoyl]methyl}pyridin-
2-ylmethylamino)acetic acid (L6H)
117.9 (d, JF–C 22.5 Hz), 115.6, 113.5, 40.8 (CH2), 27.8 (CH2). IR
(KBr) (cm−1): 1681.32 (vs); 1498.64 (s); 1446.54 (s); 1205.33 (vs).
HPLC r.t. = 10.6 min.
Picolylaminodiacetic acid (PADA) (0.897 g, 4 mmol) was
dissolved in anhydrous THF (20 mL) with the addition of
Et3N (1.7 mL, 12 mmol). To this solution, N,N-bis[2-oxo-3-
oxazolidinyl]phosphorodiamidic chloride (1.018 g, 4 mmol) was
added and the resulting mixture was stirred vigorously under
nitrogen in a water bath at 20 ◦C for 1 h and then filtered.
5.5 mL of the filtrate, which contains 1 mmol of the picolylamin-
odiacetic anhydride, was added to a solution of 6-amino-4-[(3-
bromophenyl)amino]quinazoline (3) (0.315 g, 1 mmol) dissolved
in THF (5 mL). The solution was stirred under nitrogen for 2 h
and then concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel using a mixture
of CH2Cl2–EtOH 5 : 1 to give L6H (0.350 g, 67%) as pale yellow
solid. Elem. Anal. (C24H21BrN6O3) Calc: C, 55.29%, H, 4.06%, N,
16.12%. Found: C, 54.95%, H, 4.12%, N, 16.01%; dH (500 MHz;
(CH3)2SO; Me4Si) 10.82 (1H, s, CONH), 9.97 (1H, s, NH), 8.70
(1H, s, H(5)), 8.61 (1H, d, H(6)py), 8.58 (1H, s, H(2)), 8.18 (1H, s,
H(2ꢀ)), 8.08 (1H, d, H(7)), 7.87 (1H, d, H(6ꢀ)), 7.81 (1H, H(8)),
7.79 (1H, H(4)py), 7.50 (1H, d, H(3)py), 7.35 (1H, t, H(5ꢀ)), 7.30
(1H, H(4ꢀ)), 7.29 (1H, H(5)py), 4.04 (2H, s, py-CH2N), 3.63 (2H, s,
NCH2CON), 3.53 (2H, s, NCH2COO); dC (125 MHz, (CH3)2SO)
172.9 (COOH), 169.9 (N-CH2CON), 158.3 (C2 py), 157.3 (C4),
Synthesis and characterization of [2-(3,5-dimethylpyrazol-1-
yl)ethylamino]acetic acid (L3H) and {bis[2-(3,5-dimethylpyrazol-1-
yl)ethyl]amino}acetic acid (L4H)
1.4 g (18.5 mmol) of glycine in H2O (15 mL) was added to
a solution of N-(2-bromoethyl)-3,5-dimethylpyrazole (0.745 g,
3.7 mmol) in ethanol (20 mL) and the pH adjusted to 10 by
adding 5 M NaOH. The mixture was refluxed during four days,
maintaining the pH in the range 8–10, by adding small amounts
of 5 M NaOH. After removing the solvent under vacuum, the
crude was analyzed by NMR spectroscopy. Based on the NMR
data, the crude contains approximately 80% of L3H and 20% of
L4H. This mixture was applied on a silica gel chromatography
column, and L3H andL4Hwereseparatedusingas solventMeOH–
25% NH4OH–CHCl3 and a gradient from 10 : 2 : 88 to 98 :
2 : 0.
L3H. Yield 400 mg, 2.1 mmol; 56%, 1H NMR [d, ppm, D2O]:
5.84 (s, 1H, H(4)pz), 4.21 (t, 2H, CH2), 3.43 (s, 2H, CH2CO), 3.31
(t, 2H, CH2), 2.11 (s, 3H, CH3), 2.02 (s, 3H, CH3). 13C NMR [d,
ppm, D2O (dioxane as reference)]: 173.7 (COOH), 152.7 (C(3)pz),
144.6 (C(5)pz), 108.7 (C(4)pz), 51.9 (CH2), 49.4 (CH2), 46.7 (CH2),
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153.0 (C2), 149.2 (C6 py), 146.6 (C9), 141.2 (C3ꢀ), 137.0 (C4
py), 136.6 (C6), 130.4 (C5ꢀ), 128.6 (C8), 126.8 (C7), 125.9 (C4ꢀ),
124.3 (C2ꢀ), 123.2 (C3 py), 122.6 (C5 py), 121.1 (C1ꢀ), 120.9 (C6ꢀ),
115.5 (C10), 111.4 (C5), 59.5 (py-CH2-N), 58.3 (N-CH2-CON),
55.0 (NCH2-COOH). The HPLC retention time (UV detection at
254 nm) was 15.0 min.
(m, 2H, CH2), 3.90 (m, 1H, CH2), 3.80 (m, 1H, CH2), 3.64 (d,
1H, CH2COOH), 3.47(d, 1H, CH2COOH), 2.89 (m, 4H, 2CH2),
2.41 (s 3H, Me), 2.30 (s, 3H, Me). 13C NMR [d, ppm, (Me)2SO]:
196.5 (CO), 195.4 (CO), 194.9 (CO), 177.1 (CONH), 169.1 (COO),
157.4, 153.4 (d, JF–C 240 Hz), 153.0 (C(3), pz), 152.6, 146.3, 143.7
(C(5), pz), 136.7, 136.6, 128.5, 127.0, 124.0, 123.0 (d, JF–C 21.6),
118.9 (d), 116.5 (d, JF–C 21.6Hz), 115.3, 111.6, 107.5 (C(4), pz),
62.6 (CH2), 60.6 (CH2), 55.3 (CH2), 45.3 (CH2), 30.5 (CH2), 15.4
(CH3-pz), 11.1 (CH3-pz). IR (KBr, cm−1): 2024 (s), 1917 (sh), 1892
(s, br), (1701), (1645), (1616). HPLC (UV/Vis detection 254 nm)
retention time: 19.4 min. Elem. Anal. (C29H26ClFO6N7Re.2H2O;
MW: 845.26) Calc: C, 41.21%, H, 3.58%, N, 11.60%; Found: C,
41.15%, H, 3.07%, N, 10.78%.
General procedure for preparation of rhenium complexes 7 and 8
7 mg (14 lmol) of [ReNS3(PMe2Ph)] was dissolved in 2 mL of
chloroform. After addition of 14 lmol of isocyanide L1 or L2 the
reaction mixture was refluxed for 1 h. After washing the reaction
mixture twice with water the solvent was evaporated and the
complex was purified by column chromatography on silica gel
60 using a mixture of chloroform–methanol–25% ammonia (80 :
60 : 0.2) as eluent. Recrystallization of the crude complexes from
chloroform–methanol (50 : 50, v/v) yielded complexes 7 and 8.
11. Yield 65 mg, 82%. mmax(KBr)/cm−1 2021 (s, C O), 1917 (s,
≡
≡
≡
=
C O), 1871 (s, C O), 1621 (m(C O)). dH (500 MHz; (CH3)2SO;
Me4Si) 10.68 (1H, s, CONH), 10.03 (1H, s, NH), 8.79 (1H, d,
H(6)py), 8.77 (1H, s, H(5)), 8.61 (1H, s, H(2)), 8.16 (1H, s, H(2ꢀ)),
8.15 (1H, t br, H(4)py), 7.92 (1H, d, H(7)), 7.87 (1H, m, H(6ꢀ)), 7.85
(1H, m, H(8)), 7.84 (1H, m, H(3)py), 7.60 (1H, t, H(5)py), 7.36 (1H,
t, H(5ꢀ)), 7.31 (1H, d, H(4ꢀ)), 5.30 (1H, d, J 16.2 Hz, pyCH2N), 4.78
(1H, d, J 16.2 Hz, pyCH2N), 4.78 (1H, d, J 16.2 Hz, NCH2CON),
4.55 (1H, d, J 16.2 Hz NCH2CON), 4.17 (1H, d, J 17.0 Hz,
NCH2COO), 3.82 (1H, d, J 17.0 Hz, NCH2COO); dC (125 MHz,
(CH3)2SO) 197.2 (Re-CO), 196.8 (Re-CO), 194.9 (Re-CO), 179.0
(-COORe), 166.9 (N-CH2CON), 159.6 (C5 py), 157.3 (C4), 153.1
(C2), 151.9 (C6 py), 146.6 (C9), 141.1 (C3ꢀ), 140.6 (C4 py), 136.0
(C6), 130.3 (C5ꢀ), 128.2 (C8), 127.1 (C7), 126.0 (C4ꢀ), 125.9 (C5
py), 124.4 (C2ꢀ), 124.1 (C3 py), 121.1 (C1ꢀ), 121.0 (C6ꢀ), 115.5
(C10), 111.7 (C5), 69.4 (N-CH2-CON), 68.2 (py-CH2-N), 62.0
(NCH2-COORe). HPLC (UV/Vis detection 254 nm) retention
time: 16.9 min; Elem. Anal. (C27H20BrN6O6Re) Calc. C,41.02%,
H, 2.55%, N, 10.63%; Found: C, 40.90%, H, 2.87%, N, 10.50%.
[Re(NS3)(CN-quinazoline-Cl,F)] (7). Olive-green crystals,
yield: 90%. MS (ES+): 751.45 [M + H]+. IR (KBr, cm−1): 1958 (v
1
≡
br) m(–N C). H NMR: (DMSO-d6): d (ppm) 2.8 (1H), 2.5–3.2
(12H, N(CH2CH2S)3, 5.2 (2H, CN-CH2), 7.2–8.7 (7Harom), 9.9
(NH), 10.3 (NH-C(O)). HPLC (UV/Vis detection, 254 nm)
retention time: 12.5 min. Elem. Anal. (C24H25ClFN6OReS3 MW:
750.35) Calc. C, 38.42%, H, 3.36%, N, 11.20%; S, 12.82% Found
C, 38.75%, H, 3.54%, N, 11.39%, S, 13.02%.
[Re(NS3)(CN-quinazoline-Br)] (8). Olive-green crystals, yield:
94%. MS (ES+): 777.22 [M + H]+. IR (KBr, cm−1): 1953 (v br) m(–
1
≡
N C). H NMR: (DMSO-d6): d (ppm) 2.8–3.2 (1H), 2.5–3.2 (12H,
N(CH2CH2S)3, 5.1 (2H, CN-CH2), 7.2–8.8 (8Harom), 9.9 (NH), 10.4
(NH-C(O)). HPLC (UV/Vis detection, 254 nm) retention time:
12.8 min. Elem. Anal. (C24H26BrN6OReS3 MW: 776.81) Calc. C,
37.11%, H, 3.37%, N, 10.82%; S, 12.38%; Found C, 36.95%, H,
3.54%, N, 11.10%, S, 12. 61%.
Crystallographic data†
Synthesis of Re(I) complexes 9–11
Monocrystals suitable for X-ray diffraction analysis were obtained
by slow concentration of solutions of 8 and 9 in chloroform–
methanol (3 : 1 v/v) and in H2O, respectively. The X-ray data
were collected at room temperature on a SMART-CCD diffrac-
tometer (Siemens) (complex 8) and on an Enraf-Nonius CAD4
diffractometer (complex 9), using graphite monochromatized Mo-
[NEt4]2[Re(Br)3(CO)3] (58 mg, 0.08 mmol) was reacted with L3H
(1 : 2 molar ratio) in H2O, with L5H (1 : 1 molar ratio) in
◦
MeOH–H2O for 3 h at 70 C and with L6H (1 : 1 molar ratio)
in refluxing MeOH for 2 h. The complexes 9 and 10 precipitate as
white solids from the aqueous solutions upon concentration and
cooling, while 11 was obtained after evaporation of the solvent and
recrystallization from methanol–dichloromethane (75 : 25, v/v).
˚
Ka radiation (k = 0.71073 A). Empirical absorption corrections
were made. The structures were solved by direct methods. After
anisotropic refinement of the non-hydrogen atoms, the hydrogen
positions were calculated according to ideal geometries. Most
of the calculations were carried out in the SHELXTL system
with some local modifications and WINGX software package.31
Molecular graphics were prepared using ORTEP 3.32 A summary
of the crystal data, structure solution and refinement of 8 and 9 is
given in Table 1. Selected bond distances and angles are listed in
Table 2.
9. Yield 29.8 mg, 0.06 mmol, 80%. IR (KBr, cm−1): 2021,
1
=
1883 (very broad), 1627 (m(C O)). H NMR [d, ppm, CD3OD]:
6.85 (m, 1H, NH), 6.13 (s, 1H, H(4)pz), 4.43 (d, m, 1H, CH2),
4.10 (m, 1H, CH2), 3.61 (dd, 1H, CH2), 3.53–3.45 (m, 2H, CH2),
2.46 (s, 3H, CH3-pz), 2.30 (s, 3H, CH3-pz), 2.26 (m, 1H CH2).
13C NMR [d, ppm, CDCl3]: 196.4 (CO), 196.2 (CO), 195.8 (CO),
182.8 (COO), 154.8 (C(3)pz), 144.7 (C(5)pz), 108.5 (C(4)pz), 56.6
(CH2), 51.9 (CH2), 47.8 (CH2), 15.5 (CH3-pz), 11.4 (CH3-pz).
HPLC (UV/Vis detection, 254 nm) retention time: 15.9 min. Elem.
Anal. (C12O15N3H14Re MW: 466,47) Calc. C, 30.90%; H, 3.00%;
N, 9.01%; Found: C, 30.04%; H, 2.82%; N, 8.70%.
Synthesis of the 99mTc complexes 7a and 8a
Stock solutions. EDTA solution (10 mg Na2EDTA in 1 mL
H2O, i.e. 1 mg per 100 lL). Mannitol solution (50 mg mannitol in
1 mL H2O, i.e. 5 mg per 100 lL).
10. Yield 26 mg, 0.03 mmol, 42%. 1H- NMR [d, ppm,
=
(Me)2SO]: 10.46 (s, 1H, NH(C O)), 9.95 (s, 1H, NH), 8.77 (d,
1H, H(5)), 8.55 (s, 1H, H(2)), 8.10 (m, 1H, H(7)), 7.77 (m, 3H,
H(8), H(6ꢀ), H(2ꢀ)), 7.43 (t, 1H, (H5ꢀ)), 6.22 (s, 1H, H(4)pz), 4.22
99mTc-EDTA/mannitol. Transfer 100 lL EDTA solution and
100 lL mannitol solution into a vial (i.e. 1 mg Na2EDTA, 5 mg
3218 | Dalton Trans., 2008, 3215–3225
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Table 1 Crystallographic data for complexes 8 and 9
Empirical formula
M/g mol−1
C24H28BrN6O2ReS3
794.81
273(2)
C48H60N12O22Re4
1901.88
295(2)
Temperature/K
˚
Wavelength/A
0.71073
Triclinic, P1
0.71069
¯
¯
Cryst. syst., space group
Triclinic, P1
11.3584(15)
14.756(2)
˚
a/A
7.806(15)
11.38(2)
16.23(2)
91.294(19)
91.37(3)
105.35(2)
1389(4)
˚
b/A
˚
c/A
19.845(2)
88.445(11)
88.414(10)
76.384(12)
a/◦
b/◦
c /◦
3
˚
Volume/A
3230.7(8)
Z
2
2
D
cal/Mg m−3
1.900
6.071
1.934
7.549
l/mm−1
F(000)
776
1816
Crystal size/mm
0.09 × 0.07 × 0.02
1.26 to 23.84
−8/7, −12/12, −15/18
6811/4231 [Rint = 0.0651]
98.8%
0.6 × 0.26 × 0.12
2.58 to 25.68
−13/0, −17/17, −24/24
12870/12217 [Rint = 0.0231]
99.666%
Theta range/◦
Limiting indices, hkl
Reflec. collected/unique
Completeness
Absorption correction
Tmax and Tmin
Refinement method
Data/restraints/param.
S on F2
Empirical
Psi-scan
0.374 and 0.254
Full-matrix l.s. on F2
4231/3/324
0.974
0.4212 and 0.1273
Full-matrix l.s. on F2
12217/51/783
0.970
R1, wR2 [I > 2r(I)]
R1, wR2 (all data)
Dq max, min/e A−3
0.0420, 0.1005
0.0600, 0.1147
+1.267 and −0.924
0.0451, 0.1009
0.0758, 0.1105
+1.601 and −1.558
reaction of Tc(III) with NS3 and L1 or L2 (see Scheme 2)). It is
recommended to try the preparation of 99mTc-EDTA/mannitol
without continuing with step 2 until it works well.
◦
˚
Table 2 Selected bond lengths (A) and angles ( ) for compound 8 and
molecule 1 of 9
8
9 (molecule 1)
General procedure of 99mTc complexes 7a and 8a
Re(1)–C(7)
Re(1)–N(1)
Re(1)–S(2)
Re(1)–S(1)
Re(1)–S(3)
1.944(10)
2.223(8)
2.234(3)
2.241(4)
2.249(4)
Re(1)–O(4)
Re(1)–N(3)
Re(1)–N(1)
Re(1)–C(1)
Re(1)–C(2)
Re(1)–C(3)
2.135(6)
2.226(7)
2.182(7)
1.907(10)
1.885(11)
1.926(10)
Add 0.3 mg of NS3, freshly dissolved in 100 lL water, to 99mTc-
EDTA/mannitol and transfer the solution to a vial containing
about 0.1 mg L1 or L2, dissolved in 1 mL acetonitrile. Adjust
the content of acetonitrile to half or 2/3 of the labelling mixture
volume. Flush the vial with argon. Heat the solution at 60 ◦C for
about 1 h. The labelling mixture stays clear. The radiochemical
yield is about 90% (HPLC). Purification can be done by HPLC
C(7)–Re(1)–N(1)
C(7)–Re(1)–S(2)
N(1)–Re(1)–S(2)
C(7)–Re(1)–S(1)
N(1)–Re(1)–S(1)
S(2)–Re(1)–S(1)
C(7)–Re(1)–S(3)
N(1)–Re(1)–S(3)
N(1)–Re(1)–S(3)
S(1)–Re(1)–S(3)
177.3(4)
94.2(3)
85.9(2)
96.4(3)
85.8(2)
119.76(14)
93.2(3)
84.5(3)
84.5(3)
118.30(12)
O(4)–Re(1)–N(3)
N(3)–Re(1)–N(1)
O(4)–Re(1)–N(1)
C(1)–Re(1)–C(2)
C(1)–Re(1)–C(3)
C(3)–Re(1)–C(2)
C(2)–Re(1)–O(4)
C(3)–Re(1)–N(3)
C(1)–Re(1)–N(1)
C(3)–Re(1)–O(4)
76.8(2)
84.0(3)
83.1(3)
88.7(5)
87.3(4)
87.3(4)
174.3(3)
174.6(4)
175.4(3)
98.0(4)
˚
(Jupiter Proteo, 4 lm, 90 A, 250 × 10 mm, Phenomenex;
3 mL min−1; solvent A: water with 0.1% TFA (v/v), solvent B:
acetonitrile with 0.1% TFA (v/v), gradient elution: 0–20 min 20
to 80% solvent B). Retention time: 7a, 12.3 min; 8a, 12.6 min.
Synthesis of the 99mTc complexes 9a–11a
mannitol), flush the vial with argon or nitrogen, add 20 lL SnCl2
solution (0.1 mg in methanol), immediately add 300–1500 MBq
99mTcO4−, flush the vial with argon, let it react for 15 to 20 min at
room temperature.
The radiochemical purity of 99mTc-EDTA/mannitol can be
checked using TLC silica gel strips (60 W, Merck) or ITLC strips.
99mTc-EDTA/mannitol moves to the front in water and stays at
the origin using acetone. The radiochemical purity is normally
>90%.
The 2nd step needs to be performed immediately after the 1st
step, thus the radiochemical purity of 99mTc-EDTA/mannitol can
not be checked before starting step 2, only at the same time (step 1
is the synthesis of Tc(III)EDTA (see Scheme 2); step 2 is the
In a nitrogen-purged glass vial, 100 lL of 10−4M water solution
of L3H (9a) or L5H (10a) or a methanol solution of L6H (11a)
was added to 900 lL of a solution of fac-[99mTc(OH2)3(CO)3]+ (1–
10 mCi) in PBS at pH = 7.4 (9a) or at pH = 5.5–6 (10a, 11a) The
◦
reaction mixtures were incubated at 100 C (9a, 10a) or at 70◦C
for 30 min (11a) and then analyzed by RP-HPLC (c detection).
Retention time: 9a, 16.0 min; 10a, 19.6 min; 11a, 17.2 min.
In vitro evaluation
Cell line studies. The human epidermoid carcinoma A431
cell line, kindly provided by Dr E. Mishani (Hadassah Hebrew
University, Israel) were maintained in high glucose D-MEM
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medium, with L-Glutamine, (PAA Laboratories GmbH) and
supplemented with 10% foetal bovine serum (PAA Laboratories
GmbH) and penicillin/streptomycin (PAA Labora◦tories GmbH)
100UI/100 lg per ml, in 5% CO2 incubator at 37 C. Cells were
subcultured every two or three days.
seeding, exponentially growing cells were incubated with various
concentrations of quinazoline derivatives (ranging from 1 nM
to 100 lM in 4 replicates) during 72 h. Controls consisted of
wells without drugs. The medium was removed and the cells
were incubated for 4 h in the presence of 1 mg mL−1 MTT
(Sigma) in RPMI (PAA Laboratories GmbH) without phenol red
Inhibition of phosphorylation. A431 cells (5 × 105) were seeded
in a 6-well plate and grown to 70% confluence. Then, cells were
incubated for 24 h in the presence of fresh medium without
foetal bovine serum. The day after, cells were incubated in the
presence of quinazoline derivatives at various final concentrations
ranged from 1 to 10000 nM, for 2 h and then EGF was added
(20 ng mL−1) for 5 min. Then, the medium was removed, cells
were washed with PBS and cell lysis buffer (50mM Tris-HCl
pH 7,4, 150mM NaCl, 1% Triton X-100, 1 mM PMSF, 50 lg
mL−1 approtinin, 2 mM sodium orthovanadate, 50 lg mL−1
leupeptine and 5 mM EDTA) was added. Controls consisted
of wells without quinazoline derivatives and with or without
EGF. The total amount of protein was estimated by a BCA
assay in ELISA plates, using a BSA standard curve. A volume
of lysis mixture, corresponding to 30 lg of total protein, was
loaded onto polyacrylamide gel (8%), proteins were separated
by electrophoresis and transferred to a PVDF membrane. For
visualization of molecular weight bands, the membrane was
immersed in Ponceau reagent (0.5% Ponceau in 1% acetic acid)
for 5 min. The membrane was washed in H2O, blocked overnight
in TTN buffer (10mM Tris pH 7.4, 0.2% Tween 20, 170mM NaCl)
with 5% milk (1% fat) and incubated for 2 h in antiphosphotyrosine
antibody diluted 1/2000 (PY20, Santa Cruz Biotechnology).
Then, the membrane was washed 3 times in TTN and incubated
in a horseradish peroxidase-conjugated secondary anti mouse
antibody diluted 1/3000, (Sigma) for 2 h and finally washed 3 times
in TTN. Detection was performed using a chemiluminescent
detection system according to the manufacturer’s instructions
(ECL kit, Amersham). Band quantification was performed using
the Band leader ver. 3.00 program and the percentage of EGFR
autophosphorylation inhibition was calculated.
◦
at 37 C. The MTT solution was removed and 100 mL well−1
of isopropanol was added. After thorough mixing, absorbance
of the wells was read in an ELISA reader at test and reference
wavelengths of 540 and 620 nm, respectively. The mean of the
optical density of different replicates of the same sample and
the percentage of each value was calculated (mean of the OD
of various replicates/OD of the control). The percentage of
the optical density against drug concentration was plotted on
a semilog chart and the IC50 from the dose response curve was
determined.
Results and discussion
Previous studies have shown that TcIII mixed-ligand com-
plexes with tetradentate/monodentate NS3/isonitrile coordina-
tion, [Tc(NS3)(CN-R)], are lipophilic and stable against exchange
reactions in vivo.33 These “4 + 1” organometallic Tc complexes
appear suitable to wrap the metal well in a molecule with receptor-
binding functionality.34,35
The here introduced compounds contain the (3-chloro-
4-fluorophenyl)quinazoline-4,6-diamine (2) or (3-bromophenyl)-
quinazoline-4,6-diamine (3) moiety linked to the neutral and
oxo-free technetium(III) chelate unit via a isocyano-modified
C3 alkyl amide. The synthesis of the isocyano-modified C3
alkyl amides, L1 and L2, involves the coupling of N-formyl-
b-alanin (1) to (3-chloro-4-fluorophenyl)quinazoline-4,6-diamine
(2) and (3-bromophenyl)quinazoline-4,6-diamine (3), respec-
tively, followed by the formation of the appropriate isonitrile
(Scheme 1).
In the present study rhenium complexes 7 and 8 were used
as reference compounds for complete chemical characterization,
identification of the 99mTc complexes (7a and 8a), and for in vitro
receptor binding assay. As can be seen in Scheme 2, after preparing
the precursor complex [Re(NS3)PMe2Ph] the desired “4 + 1”
In vitro growth inhibition assay. The A431 cells were seeded
in a 96-well plate at a density of 3000 cells per 100 lL per
well and incubated for 24 h for attachment. The day after
Scheme 1 Synthesis of L1 and L2.
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Scheme 2 Synthesis of MIII complexes (M = Re,7, 8; 99mTc, 7a, 8a).
complexes 7 and 8 were formed by addition of the appropriate
isocyanide ligands. The characterization of these complexes was
based on ES/MS, IR, elemental analysis, NMR and, in the case
of 8, by X-ray diffraction analysis.
An ORTEP drawing of 8 is shown in Fig. 1, and the most
relevant bond distances and angle are presented in Table 2. The
complex adopts the trigonal-bipyramidal (tbp) geometry with the
trigonal plane formed by the three thiolate sulfurs of the tripodal
ligand. The central nitrogen atom of the chelate ligand and the
monodentate isocyanide bearing the quinazoline moiety occupy
the apical positions. The Tc–Sthiolato distances are restricted in a
˚
of 1.94 A. The N1–Re–C7 axis shows only slight deviation from
linearity (177.3◦). Also the other angles of the inner coordination
sphere are consistent with the tbp arrangement.
The IR spectra of the Re reference complexes 7 and 8 exhibit a
strong andvery broad bandinthe 1950–1960cm−1 range indicating
the coordination of the isocyanide group. This band is significantly
shifted to lower energies relatively to the corresponding free
ligands L1 and L2 (D = 199 cm−1, 7; D = 202 cm−1, 8), indicating
significant p-back donation from the metal to the isonitrile
ligands.
The “4 + 1” 99mTc mixed-ligand complexes 7a and 8a were
obtained in a two-step reaction (Scheme 2). In the first step 99mTcIII-
EDTA was formed by reduction of 99mTcO4 (generator eluate)
˚
narrow range (2.234(3)–2.249(4) A) and compare well with those
−
previously reported for trigonal bipyramidal [Tc(NS3)(CN-R)]-
type complexes.33 This applies accordingly for the Re–C7 distance
in Na2EDTA solution. In the second reaction step the EDTA
was substituted by the tripodal ligand tris(2-mercaptoethyl)amine
and the appropriate monodentate isocyanide (L1, L2) resulting in
the formation of the desired “4 + 1” 99mTc complexes (7a, 8a).
The yields obtained by this procedure were between 50 and 70%.
The complexes presented high radiochemical purity after HPLC
purification. The 99mTc complexes and the analogous Re complexes
exhibited identical retention times in RP-HPLC investigations.
Previous studies have also shown that the fac-[M(CO)3]+ moiety
can be stabilized by chelators with different denticities and/or
donor atom sets.36 We and others have used mainly tridentate
chelators and we have shown the robustness of these complexes as
well as their suitability for labelling different biomolecules.24,36–43
To evaluate the effect of the metal fragment on the biological
properties of the quinazoline pharmacophore, we have synthe-
sized and characterized monoanionic and potentially tridentate
chelators with a N2O donor atom set and studied their suitability
for labelling quinazoline fragments with the fac-[M(CO)3]+ moiety.
One of the chelators has a pyrazolyl unit (L3H) and the other has
a pyridine moiety (L6H). The pyrazolyl-containing ligand L3H
was preparedby refluxingN-(2-bromoethyl)-3,5-dimethylpyrazole
with glycine in water–ethanol (1 : 1) during 4 days, while
maintaining the pH between 8 and 10. As can be seen in Scheme 3,
Fig. 1 ORTEP view of 8 with thermal ellipsoids drawn at 30% probability
level.
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Scheme 3 Synthesis of L3H.
during the formation of L3H the symmetric pyrazolyl containing
chelator L4H is also formed. The yield of L3H is maximized up
to 80% by using a five-fold excess of glycine. Compound L3H was
purified by chromatography and its characterization was based on
spectroscopic data.
Before conjugation of the novel chelator L3H to the quinazo-
line fragment, its coordination capability towards fac-[Re(CO)3]
moiety was studied. As can be seen in Scheme 4, L3H reacts with
[NEt4]2[Re(Br)3(CO)3] in water, in a 1 : 2 molar ratio, yielding the
neutral complex [Re(j3-L3)(CO)3], (9). Single crystals suitable for
X-ray crystallography were obtained by slow concentration of a
solution of the complex in water.
An ORTEP drawing of 9 is shown in Fig. 2, and the most
relevant bond distances and angles are presented in Table 2. The
complex adopts a distorted octahedral coordination geometry.
The ReI is coordinated to three carbonyls in a facial arrangement,
the three remaining coordination positions being occupied by the
oxygen atom of the carboxylate group and by the two nitrogen
atoms of the pyrazolyl ring and secondary amine.
The IR spectrum of 9 displays two very strong m(CO) bands
in the 2021–1883 cm−1 range, confirming the fac-coordination
geometry found in the solid state. The presence in this spectrum
of a very strong band at 1627 cm−1 confirms the coordination
of the oxygen atom of the carboxylic group to the metal center
−1
(Dm(C O) = 25 cm relative to the free ligand). In the 1H NMR
=
spectrum of 9 the resonances due to the pyrazolyl ring, as well
as the resonances due to the ethylenic and methylenic groups, are
downfield shifted compared to the free ligand L3H. Also, due to
the tridentate coordination mode of L3 the hydrogen atoms of
the ethylenic and methylenic groups become diastereotopic. In the
13C NMR spectrum of 9 the three resonances due to the carbonyl
groups appear at 196.4, 196.2 and 195.8 ppm, whereas the carbon
of the carboxylate appears at 182.8 ppm. The latter is significantly
downfield shifted relative to the same frequency in L3H (Dd =
9.1 ppm), in agreement with the increase found in m(CO).
At the 99mTc level, the [99mTc(j3-L3)(CO)3] complex (9a) was
obtained by reacting the fac-[99mTc(CO)3(H2O)3]+ (1a) with an
aliquot of L3H in water, at 100 ◦C for 30 min, at pH 7.4.
Complex 9a was obtained almost quantitatively (>95%) even
with very low concentration of L3H (10−5 M). The identity of 9a
(retention time (r.t.) = 16.0 min) was established by comparing
the retention time in the HPLC radiochromatogram with the
UV/Vis trace of the corresponding non-radioactive Re congener
9 (r.t. = 15.9 min). Complex 9a is stable against histidine
and cysteine challenge, and is also stable in human serum at
37 ◦C and presents low protein binding (<8%, 15 min–4 h
incubation time at 37◦C). Based on these results, a pyrazolyl-
containing chelator bearing a quinazoline fragment (L5H) was
synthesized and characterized and used to be labelled with
the technetium tricarbonyl. Compound L5H was obtained by
alkylation of the secondary amine of L3H with the quinazo-
line derivative, N-[4-[(3-chloro-4-fluorophenyl)amino]quinazolin-
6-yl]bromopropionamide (6) (Scheme 5).
Compound L5H reacts with fac-[M(X)3(CO)3]2−/+ yielding com-
plexes fac-[M(j3-L5)(CO)3] (M = Re, 10; 99mTc, 10a; Scheme 4).
Fig. 2 ORTEP view of molecule 1 of 9 with thermal ellipsoids drawn at
30% probability level.
Scheme 4 Synthesis of 9/9a and 10/10a.
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Scheme 5 Synthesis of 6 and L5H.
Unlike 9a, the functionalized complex [99mTc(j3-L5)(CO)3] (10a)
can only be obtained quantitatively (>95%) and with high specific
activity when the pH is adjusted to 5.5–6. At pH = 7.4, the value
normally used for preparing 99mTc tricarbonyl complexes, 10a was
formed but in a relatively low yield (30%), due to competitive
formation of 9a (70%). The presence of 9a is due to the hydrolysis
of L5 at the C–N bond at pH = 7.4, during the labelling reaction,
which is relatively uncommon. However, 10a when formed at a
lower pH can be left for several hours in PBS (phosphate-buffered
saline, pH 7.4, 37 ◦C), or in the presence of excess histidine or
cysteine, without any hydrolysis to 9a and/or transchelation.
As indicated in Scheme 6, compound L6H was pre-
pared by reacting picolylaminodiacetic acid with N,N-bis[2-
oxo-3-oxazolidinyl]phosphorodiamidic chloride in the presence
of triethylamine and using THF as solvent. The picoly-
laminodiacetic anhydride formed reacts with 6-amino-4-[(3-
bromophenyl)amino]quinazoline (3) in THF. The final compound
L6H was obtained after evaporation of the solvent and purification
of the crude by chromatography on silica gel. This compound
obtained in relatively high yield, was characterized by NMR,
IR spectroscopy and elemental analysis. The reactions with the
fac-[M(CO)3(H2O)3)]2−/+ yielded complexes fac-[M(j3-L6)(CO)3]
(M = Re, 11; 99mTc, 11a). The 99mTc complex 11a was found to
be stable for several hours in PBS (pH 7.4, 37 ◦C), or in the
presence of excess histidine or cysteine. The Re complexes, 10
and 11, were characterized by the normal techniques in inorganic
chemistry, and were used as surrogates for the characterization of
the analogous 99mTc complexes, 10a and 11a, by HPLC.
Cellular inhibition activities—in vitro screening
The ligands (L1, L2, L5H, L6H) as well as the rhenium complexes
(7, 8, 10, 11) were evaluated for their potency to inhibit EGFR
autophosphorylation using intact A431 cells. All the compounds
were found to be potent inhibitors, their IC50 values being below
1 lM (Table 3), indicating that they have crossed the cell membrane
(Fig. 3). So far, due to the limited number of compounds available,
a detailed structure–activity relationship is difficult to determine,
although some indications can be outlined. Functionalization
of the 6-amino group of the parent compounds (2, 3) with the
isocyano group via a C3 alkyl amide linker (L1, L2, respectively)
resulted in the most potent ligands of the series. L5H and L6H
showed lower potency compared to L1 and L2 respectively. This
can be attributed to the ionization of their COOH group that
lessens the penetration of cell membrane.
The incorporation of the rhenium cores ReNS3 and Re(CO)3
does not decrease dramatically the potency of the corresponding
ligands. Among the complexes tested compound 8 is the most
potent, even more potent than all the other ligands (L1, L5H,
L6H).
Scheme 6 Synthesis of L6H and of fac-[M(j3-L6)(CO)3] (M = Re, 11; 99mTc, 11a): i BOP-Cl, Et3N, THF, 20 ◦C; ii THF, RT.
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chelators allowed the 99mTc labeling of the quinazoline fragments
in high yield and with high radiochemical purity. The nature
and structure of the radioactive complexes were established by
comparing their HPLC profiles with those of the corresponding
Re surrogates, which were fully characterized by normal analytical
techniques, including by X-ray diffraction analysis, in the cases of
complexes 8 and 9. The radioactive complexes are quite stable to
oxidation and/or transchelation in physiological media. In vitro
studies have shown that the quinazoline fragments after conju-
gation to an isocyanide monodentate ligand or to a pyrazolyl-
containing tridentate chelator as well as the corresponding metal
complexes are still able to cross the cell membrane of A431
cells inhibiting significantly the EGFR autophosphorylation and
cell growth. These two effects were also found for the pyridine-
containing chelator and for the corresponding metal complex,
although to a lesser extent. To the best of our knowledge, the work
described in this article is the first attempt to target the intracellular
part of EGFR with a quinazoline fragment coordinated to a metal
center, through bifunctional chelators tailor-made to stabilize Re
and TcIII and TcI. Based on these findings biodistribution studies
with 99mTc complexes in healthy and tumor-bearing mice are
currently in progress to prove their usefulness as tumor imaging
agents.
Table 3 Inhibition of EGFR tyrosine kinase and A431 cell growth by
quinazolines and their rhenium complexes
Inhibition of autophosphorylation
IC50/nMa
Inhibition of cell
Compound
Growth IC50,/lMb
L1
7
130 39
481 20
4.2 0.9
6.4 1.8
2.9 0.3
2.9 1.6
2.1 1.1
7.3 1.2
23.8 5.1
41.2 13.6
4.2 0.2
4.8 0.2
L2
8
72
3
108 13
236 31
331 27
545 33
756 137
4.6 1.3
3.5 1.1
L5H
10
L6H
11
2
3
a Concentration (nM) needed to inhibit by 50% the autophosphorylation
of EGFR in A431 cells (detected by immunoblotting). b Dose-response
curves were determined at six concentrations. The IC50 values are the
concentration (lM) needed to inhibit cell growth by 50%. IC50 values
for each derivative were obtained from three independent experiments
and are represented as mean standard deviation. Each experiment was
performed in duplicate in the case of inhibition of autophosphorylation
and in quadruplicate in the case of inhibition of cell growth.
Acknowledgements
This project was part of an IAEA Coordinated Research
Project “Development of 99mTc-based small bio-molecules using
novel 99mTc cores”. Special thanks go to D. V. S. Narasimhan
and M. R. A. Pillai of the Department of Nuclear Sciences and
Applications of the IAEA, for their efforts in the success of this
Coordinated Research Project.
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