Modified PEG-anilinoquinazoline derivatives as potential EGFR PET agents

Article abstract of DOI:10.1002/jlcr.1569

Inhibition of epidermal growth factor receptor tyrosine kinase (EGFR-TK) has emerged as a major approach for cancer-targeted therapy. Consequently, there has been a great interest in the use of labeled EGFR-TK inhibitors as positron emission tomography (P

Full text of DOI:10.1002/jlcr.1569

Research Article
Received 24 September 2008,
Revised 29 October 2008,
Accepted 29 October 2008
Published online 16 December 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1569
Modified PEG-anilinoquinazoline derivatives
as potential EGFR PET agents
Ã
Samar Dissoki, Renana Eshet, Hana Billauer, and Eyal Mishani
Inhibition of epidermal growth factor receptor tyrosine kinase (EGFR-TK) has emerged as a major approach for cancer-
targeted therapy. Consequently, there has been a great interest in the use of labeled EGFR-TK inhibitors as positron
emission tomography (PET) imaging agents. Currently, the developed agents did not yield adequate PET imaging of animal
models probably due to poor solubility, rapid washout from blood, and low stability in vivo. In order to overcome these
hurdles, new derivatives of previously reported inhibitors (ML04, 2) with decreased log P and increased solubility were
designed and synthesized. These compounds (3–5) exhibited high autophosphorylation inhibitory potency with an IC50 of
5–35 nM, decreased log P’s (3.1, 3.34, and 3.45, respectively), and significantly increased solubility (630, 300, and 120 mg/mL,
respectively) relative to the previously reported parent compound 2 (log P = 3.7, solubility = 3.5 mg/mL). The labeling of
compound 5 with [18F] and compounds 3 and 4 with [11C] and [124I], respectively, involved a one-step radiosynthesis.
Compounds 3–5 were obtained with a total decay-corrected radiochemical yields of 13, 31, and 5%, respectively, and were
found to be stable in blood. The positive outcome achieved with compounds 3–5 merits further in vivo evaluation as PET
bioprobes.
Keywords: PET; EGFR; cancer; fluorine-18; imaging
Additional anti-EGFR-targeted therapies, which are currently
undergoing clinical trial, include monoclonal antibodies, such as
Introduction
panitumumab (ABX-EGF),25–28 and low-molecular-weight com-
Epidermal growth factor receptor (EGFR) is overexpressed in
many epithelial cancers such as head and neck, lung, prostate,
and kidney.1,2 Dysregulation and malfunction of EGFR are
associated with several key features of cancer, such as
autonomous cell growth, inhibition of apoptosis, increased
angiogenic potential, invasion, and metastases.3,4 Correlation
between EGFR overexpression and metastasis formation,
therapy resistance, poor prognosis, and short survival has
prompted the design and development of various anti-EGFR-
targeted therapies.5–9 Examples of FDA-approved therapies
include low-molecular-weight reversible epidermal growth
factor receptor tyrosine kinase (EGFR-TK) inhibitors, such as
gefitinib (Iressa, ZD1839; AstraZeneca, Wilmington, PA) and
erlotinib (Taceva; Genentech, San Francisco, CA).10,11 Promising
results have been obtained using gefitinib and erlotinib in pre-
clinical models of EGFR overexpressing cell lines and xeno-
grafts.12,13 Nonetheless, these results failed to reproduce in the
clinical setting since both agents appeared to be effective only
in the subset of non-small cell lung carcinoma patients.14,15
Higher response rates to gefitinib and erlotinib therapies have
been reported in patients with EGFR expressing tumors
containing well-defined activating mutations.16–20 However,
patients who initially responded to these treatments have
occasionally developed therapy resistance due to acquired
secondary mutation in the EGFR.21 The chimeric anti-EGFR
monoclonal antibody cetuximab (Erbitux; ImClone Systems, Inc.)
has yielded positive clinical results against head and neck
cancers overexpressing the EGFR and has demonstrated activity
in colon cancer regardless of tumor EGFR expression.22–24
pounds, such as the dual EGFR/HER2 inhibitor PKI-166 and the
irreversible pan-erbB inhibitor CI-1033.9,29 The moderate clinical
outcome of most approved drugs and the inconsistency
between EGFR overexpression and response to EGFR-targeted
treatment require further investigation including careful patient
selection. The lack of accurate and comprehensive measure-
ments of the phosphorylation status of EGFR in tumors
following gefitinib or erlotinib treatment poses difficulties when
interpreting clinical data, and it is actually not possible to assess
whether the poor response is indeed due to either a lack of
specific activating mutations, the absence of a survival function
of EGFR, or an insufficient long-term occupancy of the receptor
by reversible inhibitors.15,30,31 Thus, the ability to noninvasively
quantitate EGFR content in tumors would aid in selecting
patients who are likely to benefit from anti-EGFR-targeted
therapy and in monitoring such treatment.15,30 Therefore, there
has been a growing interest in the use of EGFR-TK inhibitors as
radiotracers for molecular imaging of EGFR overexpressing
tumors via nuclear medicine modalities such as positron
emission tomography (PET).15,30,32–39 Since recent pre-clinical
Cyclotron/Radiochemistry Unit, Nuclear Medicine Department, Hadassah/
Hebrew University Hospital, Jerusalem 91120, Israel
*Correspondence to: Eyal Mishani, Cyclotron/Radiochemistry Unit, Nuclear
Medicine Department, Hadassah/Hebrew University Hospital, Jerusalem 91120,
Israel.
E-mail: eyalmi@ekmd.huji.ac.il
J. Label Compd. Radiopharm 2009, 52 41–52
Copyright r 2008 John Wiley & Sons, Ltd.

S. Dissoki et al.
and clinical publications indicate that irreversible inhibitors of that it could not be easily labeled with [18F] as opposed to
the EGFR-TK appear to circumvent resistance to treatment due compound 2 and was required to be labeled with carbon-11 on
to secondary mutations, and may benefit from a larger clinical the dimethylamine moiety. Since carbon-11 has a short half-life
application,40 we and others have mainly focused on this (20 min), biological studies such as blood stability, biodistribu-
category of inhibitors.15,30,32,41,42
tion, or PET are limited to a relatively narrow time window post-
injection of the tracer. In order to afford prolonged in vitro and
in vivo measurements, we synthesized compound 4, which has a
similar structure as 3, but contains an iodine atom attached at
the aniline ring, to afford labeling with the longer-lived isotope
Results and discussion
Chemistry
[
124I] (4.2 days). In order to decrease the lipophilicity while
The main disadvantage of labeled anilinoquinazoline derivatives
developed thus far was their high log P and poor solubility,
which led to moderate tumor uptake, fast clearance from blood,
and high non-specific binding in tumor.15,30,33 Recently, in order
to circumvent these major drawbacks, an fluoro-polyethylengly-
col (FPEG) functional group was attached to the quinazoline ring
at the 7-position (compounds 1a–c and 2, Figure 1).43 While no
improvement was obtained with compounds 1a–c, compound
2 (ML04 derivative) exhibited a minor decreased lipophilicity, a
significantly improved solubility compared with the parent
compound ML04, and an irreversible binding characteristic as
opposed to 1a–c derivatives.43 Since the ML04 derivative (2)
showed promising results, we have continued the development
of this derivative in order to further improve solubility, decrease
log P, decrease blood clearance, and potentially minimize non-
specific binding in vivo.33
Three different pegylated ML04 derivatives 3–5 (Figure 2)
were designed, synthesized, and labeling routes were devel-
oped with [11C], [124I], and [18F], respectively.
Compounds 3 and 4 contain a free hydroxyl group at the end
of the polyethylenglycol (PEG) chain attached at the 7-position
of the quinazoline ring, rather than fluorinated PEG as in
compound 2. The major disadvantage with compound 3 was
enabling labeling with fluorine-18 at the end of the PEG chain in
one radiochemical step, compound 5 was synthesized (Figure
2). Compound 3 was prepared according to the synthesis shown
in Scheme 1. The synthesis of compounds 6–10 has already
been described.36,44 Compound 10 was dissolved in EtOH/H2O,
hydrazine monohydrate and Raneys nickel were added to yield
the reduced compound 11 (75%). Compound 11 was reacted
with bromo/chlorocrotonylchloride (12) at 01C to yield 13 (52%),
and the latter was reacted with dimethylamine in the presence
of N,N-diisopropylethylamine to yield 3 (28%). Compound 13
was dissolved in DMSO and reacted with monomethylamine at
01C for 15 min to obtain 14 (Scheme 1), a precursor for the [11C]
radiolabeling synthesis. The crude product 14 was kept at
ꢀ201C without any further purification.42 The synthesis of
compound 4 is shown in Scheme 2. Compound 4 was obtained
after five synthetic steps with 12% total yield.
The synthesis of compound 25, a precursor for the [124I]
radiosynthesis, is outlined in Scheme 3. Compound 8 was reacted
with 3-bromoaniline at room temperature to give 20 (54%).36
Compound 20 was reacted with 9 in the presence of potassium
trimethylsilanolate at room temperature to yield 21 (48%). The
latter was reduced to amine at 40–501C to give 22 (57%). Bromo/
chlorocrotonylchloride (12) was reacted with 22 at 01C for 1 h to
yield 23 (23%). Compound 23 was reacted with dimethylamine in
THF in the presence of N,N-diisopropylethylamine at 0–601C for
2h to yield 24 (25%). Tetrakis(triphenylphosphine) palladium(0)
and bis(tributyltin) were added to a solution of 24 dissolved in dry
toluene and refluxed for 2 h to yield 25 (28%).
N
F
Cl
Cl
F
Cl
Cl
Cl
O
O
HN
HN
HN
HN
O
The synthesis of compound 5 is outlined in Scheme 4. The
preparation of compounds 26–28 has been described pre-
viously.43 Compound 28 in THF was reacted with diethanola-
mine and DMF at 0–601C for 2 h to obtain 5 (14%). The synthesis
of compound 30, a precursor for the [18F] labeling of 5 in one
radiochemical step, is shown in Scheme 5. Compound 13 in
dichloromethane was reacted with mesyl chloride (MsCl) in the
presence of triethylamine at 01C to obtain 29 (76%). Compound
29 dissolved in THF was added to a solution of diethanolamine
N
N
O
N
N
n = 2,4, 6
4
F
F
2
1a-c
Figure 1. Chemical structures of compounds 1a–c and 2.
HO
OH
N
N
N
F
Cl
Cl
F
Cl
Cl
O
O
O
HN
HN
HN
I
HN
O
HN
O
HN
N
N
N
N
N
O
N
4
4
4
OH
3
OH
4
F
5
Figure 2. Chemical structure of ML04 derivatives 3, 4, and 5.
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J. Label Compd. Radiopharm 2009, 52 41–52

S. Dissoki et al.
OH
OH
Cl
N
OTBDMS
4
H
O2N
F
O
O2N
F
N
N
N
HNO3/H2SO4
°C/2h
SOCl2/DMF
9
°C/4h
95
100
N
KOSi(CH3)3/ 2h
F
N
7
8
6
F
Cl
Cl
F
Cl
Cl
H2NNH2.H2O/Ra-Ni
HN
HN
O2N
O
H2N
O
°C
THF/0
1h
Cl
N
N
+
Cl/Br
°C/1h
(EtOH:H2O)(9:1)/80
O
N
N
4
4
12
10
11
OH
OH
Br/Cl
N
F
Cl
Cl
F
Cl
Cl
O
O
HN
HN
HN
O
HN
N
DMA/N,N-DIPEA
THF/0-60°C/1h
N
N
O
N
4
4
13
3
OH
OH
3
2
1
5
m
i
n
NH
F
Cl
Cl
O
HN
N
HN
O
N
4
OH
14
Scheme 1.
in THF followed by the addition of DMF and stirred for 1 h at 01C 14 dissolved in a mixture of THF, DMSO, and CH3CN (with this
to obtain 30 (28%).
solvent composition the higher yield was obtained) at ꢀ151C.42
The [11C] methylation reaction was carried out at 801C for 5 min,
and the crude mixture was automatically injected onto the HPLC
preparative column to yield [11C]-3 (radiochemical purity = 98%,
decay-corrected radiochemical yield of 13%, specific activity of
2300 Ci/mmol (n = 6)) in a total radiosynthesis time of 1 h
including purification and formulation. The iodine-124 radio-
synthetic route is described in Scheme 7, [124I]-4 was obtained
with radiochemical yield of 31% (n = 3), 97% purity, and specific
activity over 6000 Ci/mmol (detection limit) in a total radio-
synthesis time of 2.5 h including purification and formulation.
Radiochemistry
The carbon-11 radiolabeling reaction to obtain compound [11C]-
3 is outlined in Scheme 6. The strategic radiolabeling approach
is based on the [11C] methylation reaction of the monomethyl-
amine group of the amide attached at the 6-position of the
quinazoline ring. The [11C] methyliodide labeling reagent was
prepared according to a previously described procedure45 and
then was distilled to a second reactor that contained precursor
J. Label Compd. Radiopharm 2009 52 41–52
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S. Dissoki et al.
Cl
N
I
I
O N
HN
N
2
CH Cl
2
2
N
+
O N
2
OTBDMS
i-PrOH/0.5h
N
H
+
O
F
NH
2
4
F
15
8
16
9
I
I
HN
N
HN
N
12
KOSi(CH )
O N
2
H N
3 3
H NNH H O/Ra-Ni
2
2
2
2
N
DMSO/2h
THF/0
°C/1h
°C/1h
35-40
O
O
4
OH
4
OH
17
18
Br/Cl
N
O
O
I
I
HN
N
HN
DMA/DIPEA
THF/0-40°C/1h
HN
HN
O
N
N
O
N
4
OH
4
OH
19
4
Scheme 2.
Scheme 8 shows the one-step [18F] labeling of compound 5. polarizable than the fluorine and the chlorine atoms, led to a
18F]-5 was obtained with a decay-corrected radiochemical yield higher lipophilicity and a lower solubility. The log P value of
[
of 5%, specific activity of 2200 Ci/mmol (n = 3), and radio- compound 5 was decreased to 3.45 and the solubility was
chemical purity of 97%. The achieved low radiochemical yield increased to 120 mg/mL (Table 1). All of the above log P values
was probably due to the reactive double bond of the were significantly better than those of ML04 (log P = 3.9,
butenamide residue attached at the 6-position of the quinazo- solubility = 0.14 mg/mL) and compound 2.
line ring. It should be noted that the identity of all labeled
compounds was confirmed by analytical HPLC co-injection with Biological evaluations
standards.
Inhibitory potency test protocol
Log P and solubility
The inhibitory potency and the extent of irreversible inhibition,
which compounds 3–5 exerted on the autophosphorylation of
the EGFR, were evaluated in A431 vulval carcinoma intact cells.
The inhibitors were incubated with intact A431 cells for 3 h, and
the degree of EGFR phosphorylation was measured either
immediately after or 8-h post-removal of the inhibitor from the
medium. As previously described,33 an 80% inhibition of
phosphorylation (or more), 8 h after removal of the inhibitor
compared with the 3-h samples, suggested that the compound
was irreversible, while a 20–80% inhibition classified the
compound as partially irreversible. The IC50’s of compounds
3–5 were 5–35 nM, a slightly decreased potency as compared
with compound 2 (Table 1). This variance could also be due to
the hydrophilic characteristic of these compounds, which might
slow their penetration into the cell. Indeed, when either a
shorter or a longer incubation time was carried out, a decreased
or an increased (only up to 3 h) affinity, respectively, was
obtained. It should be noted that their irreversible inhibitory
characteristic was successfully preserved.
As far as passive diffusion into tissues and cells is involved, the
lipophilicity of the molecule (generally denoted by log P) should
be sufficiently high to allow penetration through the cell
membrane. However, high log P’s usually lead to fast clearance
from blood, accumulation in metabolic tissue, and non-specific
binding in tumor. Therefore, since these EGFR inhibitors target
the internal TK binding domain, a log P value between 1.5 and 3
would commonly be desired. The lipophilicity of compound 3
was decreased significantly (log P = 3.1), and the solubility was
increased (solubility = 640 mg/mL) compared with the parent
compound 2 (log P = 3.7, solubility = 3.5 mg/mL)43 (Table 1).
Therefore, it is evident that the free hydroxyl polyethylenglycol
(PEG) group led to a significant decrease in lipophilicity and
increase in solubility as opposed to the minor effect of the
fluorinated polyethylenglycol (PEG) chain witnessed in com-
pound 2.43 The log P and the solubility of compound 4 were also
decreased to 3.3 and increased to 300 mg/mL, respectively
(Table 1), although to a lesser extent than in compound 3. It
appears that the iodine atom, which is more hydrophobic and
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Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 41–52

S. Dissoki et al.
HN
N
Br
HN
N
Br
KOSi(CH3)3
H
OTBDMS
4
H2NNH2.H2O/Ra-Ni
O2N
F
O2N
O
+
O
N
N
DMSO/RT/2h
°C/
(EtOH:H2O)(9:1)/40-50 1h
4
20
21
9
OH
Br/Cl
O
HN
Br
HN
Br
Cl
H2N
O
°C/1h
THF/0
HN
DMA/DIPEA
Cl/Br
N
N
+
°C/2h
THF/0-60
O
N
O
N
12
4
23
4
22
OH
OH
N
N
O
O
HN
N
Br
HN
SnBu3
HN
O
HN
Bis(tributyltin)/(Ph3P)4Pd
°C/2h
N
N
Toluene/105
O
N
4
4
24
25
OH
OH
Scheme 3.
F
Cl
Cl
F
Cl
Cl
F
Cl
Cl
HN
N
HN
HN
N
O2N
O2N
H2N
O
N
N
DAST/CH2Cl2
H2NNH2.H2O
°C/0.5h
-78-RT °C/2h
Ra-Ni/80
O
O
N
N
4
4
4
F
10
26
27
OH
F
HO
OH
N
Br/Cl
F
Cl
Cl
F
Cl
Cl
O
O
4
HN
N
HN
12
DIPEA/0°C/1h
HN
DEA/DMF/THF
0-60
HN
O
N
°C/2h
THF/
O
4
N
N
5
28
F
F
Scheme 4.
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S. Dissoki et al.
HO
OH
O
N
Br/Cl
Br/Cl
F
Cl
Cl
F
Cl
Cl
F
Cl
Cl
O
O
NH
NH
NH
NH
NH
NH
NH(CH2CH2OH)2
MsCl/Et3N
°C/1h
N
N
N
CH2Cl2/0
°C/1h
DMF/THF/0
O
N
O
N
O
N
4
4
4
13
O
29
O
S
30
OH
O
S
O
O
O
Scheme 5.
H311
C
N
NH
F
Cl
Cl
F
HN
Cl
Cl
O
O
HN
N
HN
HN
11CH3I/80°C
DMSO/THF/CH3CN
N
N
O
O
N
4
4
OH
OH
[
11C]-3
14
Scheme 6.
N
N
O
O
124I
SnBu3
HN
N
HN
HN
O
HN
Na124I/H2O2/HCl
N
N
O
N
4
4
OH
OH
25
[
124I]-4
Scheme 7.
HO
OH
HO
OH
O
N
N
F
Cl
Cl
F
Cl
Cl
O
HN
HN
K18F/Kryptofix
HN
HN
N
N
DMSO/120
°C/20 min
O
N
O
N
4
4
[
18F]-5
18F
30
O
O S O
Scheme 8.
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J. Label Compd. Radiopharm 2009, 52 41–52

S. Dissoki et al.
Table 1. IC50 values (nM) after 3-h incubation or 8-h post-incubation with A431 intact cells, log P and solubility values of ML04
and its derivatives
Compound
IC50 (nM)/3-h after incubation
(n = 4)
IC50 (nM)/8-h post-incubation
(n = 4)
Log P
(n = 3)
Solubility (mg/mL)
(n = 3)
ML04
573
372
872.5
1571
3576.5
2575
1577
2075
79714
95710
3.970.4
3.770.2
3.170.88
3.3470.96
3.4570.8
0.1470.56
3.570.12
63070.78
30070.33
12070.48
2
3
4
5
(v/v) ammonium formate 0.1 M:acetonitrile for the semi-pre-
parative column, 50:48:2 (v/v/v) ammonium formate 0.1
M:acetonitrile:THF for the preparative column, 55:45 (v/v)
acetate buffer:acetonitrile, and 53:47 (v/v) ammonium formate
0.1 M:acetonitrile for the analytical C-18 columns. 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 (GE) were used. Specific radio-
activities were determined by HPLC, using cold mass calibration
curves. Radiosyntheses, using fluorine-18 and carbon-11, were
carried out on [18F] and [11C] GE modules. Fluorine-18 was
produced on an IBA 18/9 cyclotron by irradiation of 2 mL water
target (97%-enriched [18O]water) by the [18O(p,n)18F] nuclear
reaction. [11C]CO2 was produced by the [14N(p,a)11C] nuclear
reaction on nitrogen containing 0.5% oxygen. [124I]-NaI was
purchased as a 0.02 M solution from Ritverc GmbH, Germany.
A431 human epidermoid vulval carcinoma cells were grown in
Dulbeccco’s modified Eagle medium (Biological Industries, Beit
Haemek, Israel) supplemented with 10% fetal calf serum and
antibiotics (penicillin 105 units/L, streptomycin 100 mg/L) at
371C, 5% CO2.
80
70
60
50
40
30
20
10
0
[11C]-3
[124I]-4
[18F]-5
Compound
0 min 30 min 60 min 90 min 120 min
Figure 3. Extraction intact values of compounds [11C]-3, [124I]-4, and [18F]-5 in
blood stability assay at different time periods (n = 3).
In vitro stability in blood
In vitro blood stability was determined by incubating the
radioligands, [11C]-3, [124I]-4, and [18F]-5, in human blood and
plasma at 371C for various time periods. Blood samples were
extracted with THF/CH3CN (30:70) (v/v) at different time points
(Figure 3). The extracted radioactivity was measured and loaded
onto reversed-phase TLC plates and radioactive bands were
visualized by a phosphor image plate. No radioactive degrada-
tion products were observed in blood and plasma by a
phosphor image plate during these different time points.
Chemistry
Compounds 6–10, 20, and 26–28 were prepared according to
the literature procedures.36,43
2-[2-(2-2-[6-Amino-4-(4,5-dichloro-2-fluoro-phenylamino)-quinazo-
lin-7-yloxy]-ethoxy-ethoxy)-ethoxy]-ethanol (11)
Hydrazine monohydrate (0.71 mmol, 34.5 mL) and Raneys nickel
solution (0.8 mL) were added to a solution of compound 10
(0.183 g, 0.35 mmol) dissolved in EtOH/H2O (9:1) (25 mL). The
reaction mixture was stirred at 801C for 30 min. The solution was
cooled and filtered over Celites; the filtrate was evaporated
under reduced pressure, and the residue was extracted with
dichloromethane ( ꢁ 3), washed with brine ( ꢁ 1), dried with
Experimental
General
All chemicals were purchased from Sigma-Aldrich, Fisher
Scientific, Merck, or J. T. Baker. THF was refluxed over sodium/
benzophenone and dichloromethane was refluxed over phos-
phorous pentoxide (P2O5). Other solvents were purchased as
anhydrous. 1H-NMR spectra were recorded on 300 MHz spectro-
meters in DMSO-d6. 1H signals are reported in ppm. 1H-NMR
signals are referenced to either the residual proton (2.50 ppm for
DMSO-d6; 7.25 ppm for CDCl3) of a deuterated solvent or TMS as
the internal standard. Mass spectra were obtained on a
spectrometer equipped with CI, EI, and FAB probes and on a
spectrometer equipped with an ESI probe. HRMS results were
obtained on MALDI-TOF and ESI mass spectrometers. Elemental
analysis was performed on a Perkin-Elmer 2400 series II analyzer
at the Hebrew University Microanalysis Laboratory. Reversed-
phase HPLC: analytical, semi-preparative, and preparative
columns C-18 mBondapaks Waters using the eluents 53:47
1
sodium sulfate, and evaporated to afford 11 (0.139 g, 75%). H-
NMR (DMSO-d6): d 9.27 (s, 1H), 8.26 (s, 1H), 7.94–7.97 (m, 1H),
7.73–7.76 (m, 1H), 7.27 (s, 1H), 7.1 (s, 1H), 5.39 (s, 2H), 4.58 (m,
1H), 4.28 (m, 2H), 3.86 (m, 2H), 3.25–3.62 (m, 11H). MS (m/z)
515.67 (MH1). HRMS (EI): calcd. for C22H25Cl2FN4O5: 515.1293,
found: 515.1264. M.p. = 115–1161C.
4-Bromo/chloro-but-2-enoic acid[4-(4,5-dichloro-2-fluoro-phenyla-
mino)- 7 - (2-2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy-ethoxy)-quina-
zolin-6-yl]-amide (13)
Compound 11 (0.118 g, 0.22 mmol) was dissolved in dry
THF (3 mL), and added dropwise to
a
solution of
J. Label Compd. Radiopharm 2009 52 41–52
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S. Dissoki et al.
4-bromo/chloro-but-2-enoyl chloride (12)36 (0.151 g, 0.91 mmol) 2-[2-(2-2-[4-(3-Iodo-phenylamino)-6-nitro-quinazolin-7-yloxy]-ethoxy-
ethoxy)-ethoxy]-ethanol (17)
in THF (1.5 mL) at 01C and stirred for 1 h. The solvent was
evaporated and the residue was purified by silica gel
chromatography and eluted with 5% MeOH in CH2Cl2 to obtain
Compound 16 (0.496 g, 1.21 mmol), 2-[2-(tert-butyl-dimethyl-
silanyloxy)-ethoxy]-ethanol (9) (0.561 g, 1.81 mmol), and potas-
sium trimethylsilanolate (0.466 g, 3.63 mmol) were dissolved in
dry DMSO (25 mL) and stirred for 1.5 h at room temperature.43
The crude product was extracted with ethylacetate (ꢁ 4) and
water, washed with NaHCO3 (4%) and brine (ꢁ 1), dried
(Na2SO4), and evaporated under reduced pressure. The residue
was purified on silica gel chromatography, and eluted with 3%
1
13 (0.079 g, 52%). H-NMR (CDCl3): d 9.19 (s-br, 1H), 9.14 (s, 1H),
8.8 (d, J = 9 Hz, 1H), 8.71 (s, 1H), 7.32 (s, 1H), 7.29 (s, 1H),
7.08–7.15 (m, 1H), 6.47–6.52 (m, 1H), 4.39 (m, 3H), 3.99–4.03 (m,
3H), 3.61–3.75 (m, 11H). MS (m/z) 617.6/663.27 (MH1). HRMS
(EI): calcd. for C26H28Cl3FN4O6: 617.1158/661.0627, found:
617.1137/661.0632.
1
MeOH in CH2Cl2 to obtain 17 (0.608 g, 48%). H-NMR (CDCl3): d
4-Methylamino-but-2-enoic acid[4-(4,5-dichloro-2-fluoro-phenyla-
mino)- 7 - (2-2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy-ethoxy)-quina-
zolin-6-yl]-amide (14)
8.76 (s, 1H), 8.53 (s, 1H), 8.22 (s-br, 1H), 7.71–7.73 (m, 1H), 7.64 (s-
br, 1H), 7.53–7.56 (m, 1H), 7.37 (s, 1H), 7.16 (t, J = 7.8 Hz, 1H),
4.34–4.37 (m, 2H), 3.95–3.98 (m, 2H), 3.56–3.8 (m, 12H). MS (m/z)
585.2 (MH1). HRMS (EI): calcd. for C22H25IN4O7: 585.0820, found:
585.0846.
Methylamine (2 mol/L in THF, 1 mL) was added to a solution of
compound 13 (0.01 g, 0.015 mmol) in DMSO (70 mL) at 01C. After
a 15-min reaction, NaOH (0.015 mol/L, 10 mL) was added and the
solution was vortexed for 3 min. The solution was passed
2-[2-(2-2-[6-Amino-4-(3-iodo-phenylamino)-quinazolin-7-yloxy]-ethoxy-
through 2 ꢁ C-18 cartridges (Waters Sep-Pak Plus), preactivated ethoxy)-ethoxy]-ethanol (18)
with 10 mL EtOH and 20 mL of sterile water, and dried under a
Compound 18 was prepared from 17 (0.453 g, 0.77 mmol)
stream of nitrogen. The Sep-Pak columns were eluted with THF
(5 mL) and the eluent was dried with Na2SO4 and filtered with a
0.45-mm filter. THF evaporation under nitrogen stream yielded
the crude product 14 (470% purity). MS (m/z) 612.47 (MH1).
similarly to 11 (0.173 g, 23%). 1H-NMR (CDCl3): d 8.58 (s, 1H), 8.15
(s, 1H), 7.71–7.73 (m, 1H), 7.43–7.46 (m, 1H), 7.18 (s, 1H), 7.1–7.3
(m, 1H), 7.01 (m, 1H), 6.91 (s, 1H), 4.32–4.35 (m, 2H), 3.93–3.96
(m, 2H), 3.6–3.76 (m, 12H). MS (m/z) 555.27 (MH1). HRMS (EI):
calcd. for C22H27IN4O5: 555.1120, found: 555.1104.
4-Dimethylamino-but-2-enoic acid[4-(4,5-dichloro-2-fluoro-pheny-
lamino) - 7 - (2 -2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy-ethoxy)-qui-
nazolin-6-yl]-amide (3)
4-Bromo/chloro-but-2-enoic acid[7-(2-2-[2-(2-hydroxy-ethoxy)-
ethoxy]- ethoxy-ethoxy) - 4 - (3 -iodo-phenylamino)-quinazolin-6-yl]-
Compound 13 (0.128 g, 0.193 mmol) dissolved in dry THF (2 mL) amide (19)
was added dropwise to a cooled solution of dimethylamine
Compound 19 was prepared from 18 (0.111 g, 0.2 mmol)
(2 mL) in THF (2 M), then diisopropylethylamine (0.38 mmol,
67.3 mL) was added. The reaction mixture was stirred at 01C for
30 min, and heated to 601C for an additional 30 min. The solvent
was evaporated, extracted with ethylacetate (ꢁ 3), washed with
a NaHCO3 solution (4%) (ꢁ 1), brine (ꢁ 1), dried over Na2SO4,
filtered, and evaporated. The crude product was purified by
silica gel chromatography and eluted with 15% MeOH in CH2Cl2
to obtain 3 (0.034 g, 28%). 1H-NMR (CDCl3): d 9.26 (s-br, 1H), 9.16
(s, 1H), 8.8 (d, J = 6 Hz, 1H), 8.71 (s, 1H), 7.63 (s-br, 1H), 7.32 (s,
1H), 7.02–7.07 (m, 1H), 6.41–6.46 (m, 1H), 4.37–4.39 (m, 2H),
3.98–4 (m, 2H), 3.59–3.76 (m, 12H), 3.2 (m, 2H), 2.33 (s, 6H). MS
similarly to 13 (0.038 g, 27%). 1H-NMR (CDCl3): d 9.23 (s-br,
1H), 9.07 (s, 1H), 8.65 (s, 1H), 8.2 (s-br, 1H), 7.74 (m, 2H), 7.43–7.45
(m, 1H), 7.09 (s, 1H), 7.07–7.13 (m, 1H), 6.47–6.52 (m, 1H), 4.36
(m, 3H), 4 (m, 3H), 3.59–3.74 (m, 12H). MS (m/z) 657.67/701.53.
HRMS (EI): calcd. for C26H30BrClIN4O6: 657.0955/701.0475, found:
657.0977/701.0472.
4-Dimethylamino-but-2-enoic acid[7-(2-2-[2-(2-hydroxy-ethoxy)-
ethoxy]-ethoxy-ethoxy) - 4 - (3 - iodo-phenylamino)-quinazolin-6-yl]-
amide (4)
(m/z) 626.73 (MH1). HRMS (EI): calcd. for C28H34Cl2FN5O6: Compound 4 was prepared from 19 (0.038 g, 0.054 mmol)
626.1929, found: 626.1948. M.p. = 120–1211C.
similarly to 3 (0.11 g, 32%). 1H-NMR (CDCl3): d 9.3 (s-br, 1H), 9.09
(s, 1H), 8.65 (s, 1H), 8.2 (s-br, 1H), 7.74 (m, 2H), 7.43–7.76 (m, 1H),
7.22 (s, 1H), 7–7.06 (m, 1H), 6.41–6.46 (m, 1H), 4.34–4.36 (m, 2H),
3.97 (m, 2H), 3.57–3.75 (m, 12H), 3.16–3.18 (m, 2H). MS (m/z)
666.33 (MH1). HRMS (EI): calcd. for C28H36IN5O6: 666.1805,
found: 666.1789.
(7-Fluoro-6-nitro-quinazolin-4-yl)-(3-iodo-phenyl)-amine (16)
3-Iodo-aniline (15) (0.639 g, 2.9 mmol) dissolved in isopropanol
(13 mL) was added dropwise to a solution of compound 8 (0.6 g,
2.65 mmol) in CH2Cl2 (5 mL), and stirred at room temperature for
30 min.36 The precipitation was completed by the addition of hexane;
the crystals were filtered, dissolved with methanol, and neutralized
with triethylamine. The orange solution was diluted with water to
yield a yellow precipitate, which was filtered and washed with water.
The yellow crystals were extracted with ethylacetate and washed with
NaHCO3 solution (4%) (ꢁ 3) and brine (ꢁ 1). The organic phase was
dried over sodium sulfate, filtered, and evaporated under reduced
pressure to yield 16 (0.496 g, 45%). 1H-NMR (CDCl3): d 8.85 (s, 1H), 8.79
(d, J= 7.5 Hz, 1H), 8.21 (m, 1H), 7.76 (s, 1H), 7.68–7.72 (m, 1H),
7.55–7.61 (m, 2H), 7.19 (t, J= 7.8 Hz, 1H). MS (m/z) 411.13 (MH1).
HRMS (EI): calcd. for C14H8FIN4O2: 410.9709, found: 410.9754.
M.p. = 160–1611C.
2-[2-(2-2-[4-(3-Bromo-phenylamino)-6-nitro-quinazolin-7-yloxy]-ethoxy-
ethoxy)-ethoxy]-ethanol (21)
Compound 20 (0.512 g, 1.41 mmol), 2-[2-(tert-butyl-dimethyl-
silanyloxy)-ethoxy]-ethanol (9) (0.654 g, 2.1 mmol), and potas-
sium trimethylsilanolate (0.543 g, 4.23 mmol) were dissolved in
dry DMSO (30 mL) and stirred at room temperature for 2 h. The
solution was extracted with EtOAc (4) and water, washed with
sodium bicarbonate (4%) and brine ( ꢁ 1), dried (Na2SO4), and
evaporated under reduced pressure. The residue was purified by
silica gel chromatography and eluted with 3% MeOH in CH2Cl2
1
to obtain 21 (0.364 g, 48%). H-NMR (CDCl3): d 8.77 (s, 1H), 8.55
www.jlcr.org
Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 41–52

S. Dissoki et al.
(s, 1H), 8.1 (s-br, 1H), 7.64–7.71 (m, 2H), 7.26–7.35 (m, 3H), stirred at 01C for 1 h, and heated to 601C for an additional 1 h.
4.33–4.36 (m, 2H), 3.94–3.97 (m, 2H), 3.55–3.8 (m, 12H). MS (m/z) The solvent was evaporated, and the residue was extracted with
537.93 (MH1). HRMS (EI): calcd. for C22H25BrN4O7: 537.1004, EtOAc ( ꢁ 2), washed with NaHCO3 (ꢁ 1) and brine (ꢁ 1), dried
found: 537.0985.
over Na2SO4, filtered, and evaporated. The crude product was
purified by silica gel chromatography and eluted with 7% MeOH
1
2-[2-(2-2-[6-Amino- 4 -(3-bromo-phenylamino)-quinazolin-7-yloxy]- in CH2Cl2 to afford 5 (0.0125 g, 14%). H-NMR (CDCl3): d 9.12 (s,
ethoxy-ethoxy)-ethoxy]-ethanol (22)
1H), 8.91 (s, 1H), 8.7 (m, 2H), 7.86 (s-br, 1H), 7.31 (s, 1H), 7 (m, 1H),
6.54–6.59 (m, 1H), 4.6–4.63 (m, 1H), 4.44–4.47 (m, 1H), 4.34–4.37
(m, 2H), 3.98–4 (m, 2H), 3.55–3.76 (m, 14 H), 3.42 (m, 2H),
2.71–2.74 (m, 4H). MS (m/z) 688.6 (MH1). HRMS (EI): calcd. for
C30H37Cl2F2N5O7: 688.2108, found: 688.2116.
Compound 22 was prepared from 21 (0.18 g, 0.33 mmol)
similarly to 11 (0.095 g, 57%). 1H-NMR (CDCl3): d 8.56 (s, 1H),
8.03 (s, 1H), 7.63–7.66 (m, 1H), 7.36–7.38 (m, 1H), 7.22 (m, 1H),
7.11 (s, 1H), 6.97 (s, 1H), 4.25–4.28 (m, 2H), 3.89–3.92 (m, 2H),
3.59–3.76 (m, 12H). MS (m/z) 507.87 (MH1). HRMS (EI): calcd. for
C22H27BrN4O5: 507.1218, found: 507.1243.
Methanesulfonic acid 2-[2-(2-2-[6-(4-bromo/chloro-but-2-enoyla-
mino)-4-(4,5-dichloro-2-fluoro-phenylamino)-quinazolin-7-y-yloxy]-
ethoxy-ethoxy)-ethoxy]-ethyl ester (29)
4-Bromo / chloro - but 2-enoic acid[4-(3-bromo-phenylamino)-7-(2-
2 - [2 - (2 - hydroxy-ethoxy)-ethoxy]-ethoxy-ethoxy)-quinazolin-6-yl]-
amide (23)
Methanesulfonyl chloride (0.12 mmol, 9.2 mL) was added to a
cooled solution of compound 13 (0.02 g, 0.03 mmol) in dry
CH2Cl2 (1.8 mL) and triethylamine (0.12 mmol, 21 mL). The
reaction mixture was stirred at 01C for 1.5 h, and extracted from
CH2Cl2 ( ꢁ 2) and water, washed with brine ( ꢁ 1), dried, and
evaporated. Compound 29 was obtained with 95% chemical
purity (0.017 g, 76%) without any further purification. 1H-NMR
(CDCl3): d 9.18 (s-br, 1H), 9.01 (s, 1H), 8.6 (s, 1H), 8.12 (s-br, 1H),
7.68 (m, 2H), 7.35–7.42 (m, 1H), 7.02 (s, 1H), 6.92–6.98 (m, 1H),
6.4–6.45 (m, 1H), 4.29 (m, 3H), 3.98 (m, 3H), 3.55–3.7 (m, 12H),
2.98 (s, 3H). MS (m/z) 695.73/741.2 (MH1). HRMS (EI): calcd. for
C27H30BrCl3FN4O8S: 694.3584/740.8623, found: 694.3577/
740.8630.
Compound 23 was prepared from 22 (0.215 g, 0.42 mmol)
1
similarly to 13 (0.063 g, 23%). H-NMR (CDCl3): d 9.61 (s-br, 1H),
9.13 (s, 1H), 8.63 (s, 1H), 8.03 (s, 1H), 7.69–7.77 (m, 2H), 7.28–7.4
(m, 2H), 7.09–7.14 (m, 1H), 6.57 (m, 1H), 4.33 (m, 2H), 3.88 (m,
2H), 3.62–3.77 (m, 14H). MS (m/z) 611.53/653.87 (MH1). HRMS
(EI): calcd. for C26H30Br2ClN4O6: 609.1120/653.0616, found:
609.1115/653.0610.
4-Dimethylamino-but-2-enoic acid[4-(3-bromo-phenylamino)-7-(2-
2 - [2 - (2 - hydroxy-ethoxy)-ethoxy]-ethoxy-ethoxy)-quinazolin-6-yl]-
amide (24)
Compound 24 was prepared from 23 (0.063 g, 0.096 mmol)
Methanesulfonic acid 2-[2-(2-2-[6-4-[bis-(2-hydroxy-ethyl)-amino]-
1
similarly to 3 (0.015 g, 25%). H-NMR (CDCl3): d 9.46 (s-br, 1H), but-2-enoylamino-4-(4, 5 -dichloro-2-fluoro-phenylamino)-quinazo-
lin-7-yloxy]-ethoxy-ethoxy)-ethoxy]-ethyl ester (30)
9.08 (s, 1H), 8.65 (s, 1H), 8.1 (s, 1H), 7.97 (s-br, 1H), 7.66–7.7 (m,
1H), 7.2–7.24 (m, 2H), 6.98–7.07 (m, 1H), 6.43–6.49 (m, 1H),
4.35–4.38 (m, 2H), 3.96–3.99 (m, 2H), 3.58–3.77 (m, 12H),
3.15–3.17 (m, 2H), 2.32 (s, 6H). MS (m/z) 619.33 (MH1). HRMS
(EI): calcd. for C28H36BrN5O6: 618.1905, found: 618.1927.
Compound 29 (0.017 g, 0.023 mmol) was dissolved in dry THF
(0.5 mL), and added dropwise to
a cooled solution of
diethanolamine (0.46 mmol, 44 mL) in 0.2 mL dry THF, followed
by the addition of DMF (0.092 mmol, 7 mL). The reaction mixture
was stirred at 01C for 1 h. The solvent was evaporated and the
residue was extracted with EtOAc ( ꢁ 2), washed with sodium
bicarbonate solution (4%) ( ꢁ 1) and brine ( ꢁ 1), dried (Na2SO4),
and evaporated. The residue was purified by silica gel
chromatography and eluted with 15% MeOH in CH2Cl2 to
4-Dimethylamino-but-2-enoic acid[4-(3-tributylstannyl-phenylami-
no) - 7 - (2-2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy-ethoxy)-quinazo-
lin-6-yl]-amide (25)
Compound 24 (0.05 g, 0.08 mmol) was dissolved in dry toluene
(3 mL), and bis(tributyltin) (0.32 mmol, 163.5 mL) was added,
followed by the addition of (PPh3)4Pd (0.028 g, 0.024 mmol).46
The reaction mixture was refluxed for 2 h, and the solvent was
evaporated under reduced pressure. The crude product was
purified by silica gel chromatography and eluted with 10%
MeOH in CH2Cl2 to obtain 25 (0.0183 g, 28%). 1H-NMR (CDCl3): d
9.31 (s-br, 1H), 9.12 (s, 1H), 8.6 (s, 1H), 7.74–7.77 (m, 1H), 7.68 (m,
1H), 7.59 (s-br, 1H), 7.32–7.39 (m, 2H), 6.98–7.08 (m, 1H),
6.42–6.48 (m, 1H), 4.37 (m, 2H), 3.98 (m, 2H), 3.58–3.75 (m, 12H),
3.15–3.17 (m, 2H), 2.35 (s, 6H), 0.89–1.63 (m, 27H, Bu3). MS (m/z)
829.27 (MH1). HRMS (EI): calcd. for C40H63N5O6Sn: 828.5412,
found: 828.5422.
1
obtain 30 (0.005 g, 28%). H-NMR (CDCl3): d 9.14–9.17 (m, 1H),
8.71–8.8 (m, 2H), 8.36 (s-br, 1H), 7.65 (m, 1H), 7.28–7.32 (m, 2H),
7.03–7.08 (m, 1H), 6.22–6.27 (m, 1H), 4.28–4.41 (m, 5H), 3.99 (m,
3H), 3.65–3.75 (m, 13H), 3.47 (m, 2H), 3.06 (s, 3H), 2.97–2.73 (m,
3H). MS (m/z) 764.87 (MH1). HRMS (EI): calcd. for
C31H40Cl2FN5O10S: 763.2514, found: 763.2509.
Radiochemistry
[11C]4-dimethylamino-but-2-enoic acid[4-(4,5-dichloro-2-fluoro-phe-
nylamino) - 7 - ( 2 - 2 - [ 2 -(2-hydroxy-ethoxy)-ethoxy]-ethoxy-ethoxy)-
quinazolin-6-yl]-amide ([11C]-3)
[11C]CO2 was trapped at ꢀ1601C, and the activity was
transferred into the first reactor, which contained 0.3 mL of
0.25 N LiAlH4 (0.5 mL/1.5 mL THF) at ꢀ501C. The solvent was
removed under reduced pressure, followed by the addition of HI
(57%) at 1601C. The resultant [11C]MeI was distilled through an
NaOH column to the second reactor, containing a solution of
the monomethylamine precursor (14) (8–10 mg) dissolved in a
4-[Bis-(2-hydroxy-ethyl)-amino]-but-2-enoic acid[4-(4,5-dichloro-2-
fluoro-phenyla-mino) - 7 - (2 - 2-[2-(2-fluoro-ethoxy)-ethoxy]-ethoxy-
ethoxy)-quinazolin-6-yl]-amide (5)
Compound 28 (0.087 g, 0.13 mmol) was dissolved in dry THF
(0.5 mL) and added to a solution of 2-(2-hydroxy-ethylamino)-
ethanol (5.24 mmol, 0.5 mL) in THF (1 mL), followed by the
addition of DMF (0.52 mmol, 40.8 mL). The reaction mixture was
J. Label Compd. Radiopharm 2009 52 41–52
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

S. Dissoki et al.
mixture of 0.3 mL DMSO, 0.2 mL THF, and 0.1 mL CH3CN at 4 mL water, then eluted with 1 mL EtOH and 9 mL saline. [18F]-5
ꢀ151C. The reaction mixture was heated to 801C for 5 min. THF was obtained with a 5% decay-corrected radiochemical yield,
and acetonitrile were removed under flow of argon at 801C, 97% radiochemical purity, and specific activity of 2200 Ci/mmol
then the reactor was cooled to 401C, followed by the addition of (n = 3).
1.4 mL CH3CN/H2O 50:50 (v/v). The crude product was injected
to the HPLC preparative column (Nucleosil C-18, 7 mm, Partition coefficient determination
250 ꢁ 16 mm), eluted with ammonium formate 0.1 M:CH3CN:THF
50:48:2 (v/v/v), flow = 8 mL/min. The labeled product was
Partition coefficients were measured by mixing compounds 3–5
(3 mg) with 1 mL of 1-octanol and 5 mL of phosphate buffer
collected (Rt = 20 min) in a flask containing 50 mL of NaOH
(1 M) in 70 mL water. The solution was passed though a 2 ꢁ C-
0.1 M, pH 7.4, in a test tube.47,48 The test tube was vortexed for
2 min at room temperature, followed by centrifugation for
18 cartridge (preactivated with 10 mL EtOH and 20 mL of water),
10 min. Aliquots of 50 mL were taken from the organic phase and
the buffer, dissolved in 0.45 mL acetonitrile, and injected to the
HPLC (C-18 analytical column, acetate buffer 0.1 M, pH 3.8/
then the Sep-Pak was washed with water (4 mL). The product
was eluted with EtOH (1 mL) and saline (9 mL), and collected into
the product vial. [11C]-3 was obtained after a total radio-
CH3CN 55:45 (v/v), flow = 1 mL/min). The partition coefficient
synthesis time of 1 h, 13% decay-corrected radiochemical yield
was determined by calculating the ratio of the organic to the
(EOB), 98% radiochemical purity, and specific activity of 2300 Ci/
aqueous phases.
mmol (n = 6).
Water solubility determination
[
124I]4-dimethylamino-but-2-enoic acid[7-(2-2-[2-(2-hydroxy-ethoxy)-
ethoxy]- ethoxy-ethoxy) - 4 - (3 - iodo-phenylamino) - quinazolin- 6 -yl]-
One milligram of each of the compounds (3–5) was added into
two separate test tubes (totally six tubes). Into one of the two
tubes, DMSO (1 mL) was added and, to the other, MOPS buffer
pH 7.4 (1 mL) was added.49 The test tubes were sonicated at
room temperature for 15 min, and filtered through 0.45-mm
filters. The DMSO solution was diluted ten-folds with DMSO.
Fifty microliters of each solution was injected onto HPLC system
(C-18 analytical, acetate buffer 0.1 M, pH 3.8/CH3CN 55: 45 (v/v),
flow = 1 mL/min). The solubility was calculated according to the
equation
amide ([124I]-4)
A total of 0.1 mL of H2O2 (3%) was added to a mixture of the
tributyltin derivative (25) (2 mg/0.2 mL EtOH), 20 mL of [124I]NaI
(0.02 M NaOH), and 0.1 mL of HCl (1 N) in a v-vial. The reaction
mixture was stirred at room temperature for 10 min and
terminated by the addition of 0.1 mL Na2S2O5 (10%). The
solution was neutralized with saturated NaHCO3 (4 mL), passed
though a C-18 cartridge, and eluted with THF (2 mL). The solvent
was evaporated to a volume of 0.5 mL, under a stream of argon,
followed by the addition of 0.5 mL CH3CN/H2O 50:50 (v/v), and
injected to the HPLC (semi-preparative column, Nucleosil 100-C-
18, 7 mm, 250 ꢁ 10 mm), eluted with ammonium formate
0.1 M:acetonitrile 53:47 (v/v), flow = 6 mL/min. The labeled
product was collected (Rt = 32 min) in a flask, diluted with
0.137 mL NaOH (1 M) in 195 mL water, passed through a C-18
cartridge, and eluted with 1 mL of EtOH. The final solution of
Solubility ¼ ððpeak areabufferÞ=ðpeak areaDMSO ꢁ 10ÞÞ
ꢁ ð1000 mg=mLÞ
Biology
Irreversibility test protocol
[
[
124I]-4 was prepared from 0.09 mL EtOH and 0.91 mL of saline.
124I]-4 was obtained after a total radiosynthesis time of 2.5 h, The inhibitory potency and the extent of irreversible inhibition,
31% decay-corrected radiochemical yield, and 100% radio- which compounds 3–5 exerted on the phosphorylation of the
chemical purity.
EGFR, were evaluated in A431 vulval carcinoma cells. A431 cells
(1 ꢁ 105 cells/well) were grown in six-well plates for 48 h, and
[18F]4-[bis-(2-hydroxy-ethyl)-amino]-but-2-enoic acid[4-(4,5-dichloro- further maintained in serum-free media for an additional 18 h.
2-fluoro-phenylamino) - 7 - ( 2 - 2 - [ 2-(2-fluoro-ethoxy)-ethoxy]-ethoxy-
Duplicate sets of cells were incubated with increasing concen-
trations of compounds 3–5 (0.05% DMSO, 0.1% EtOH) for 3 h,
after which the inhibitors were removed, and a serum-free
medium was added to the wells. One set of cells was stimulated
with EGF (20 ng/mL) immediately after the removal of the
inhibitor, while the other set of cells underwent EGF stimulation
only 8 h after the removal of the inhibitor from the medium and
successive rinsing with phosphate buffer saline. Cell lysates were
prepared and loaded onto SDS-PAGE (8% acryl amide) for
Western Blot analysis, and the extent of EGFR phosphorylation
was evaluated by measuring the signal intensity of the
corresponding phosphotyrosine band using a mix of anti-
phosphotyrosine antibodies, PY20 (Santa Cruz Biotechnology
Inc.) and 4G10 (produced from Su4G10 hybridoma cells).
ethoxy)-quinazolin-6-yl]-amide [18F]-5
[18O]H2O/18Fꢀ was trapped and then transferred to a reactor
through an ion exchange column (UKE F18, Macherey-Nagel,
preactivated with 0.8 mL EtOH and 3 mL HPLC grade water) and
eluted with 0.5 mL K2CO3 (5 mg/1 mL). Kryptofix222 (18 mg/1 mL
CH3CN) was added, and the solvent was removed by an
azeotropic distillation at 951C under reduced pressure for 3 min.
A solution of mesylate precursor 30 (5 mg/0.4 mL DMSO) was
added to the reactor containing the dried complex K18F-
kryptofix. The solution was heated to 1201C for 20 min, then it
was cooled to 351C, and 1.6 mL of CH3CN/H2O 50:50 (v/v) was
added to the reactor, and injected to the HPLC preparative
reversed-phase column (Nucleosil 100-C-18, 250 ꢁ 16 mm),
eluted with ammonium formate 0.1 M/acetonitrile/THF
(50:48:2) (v/v/v), flow = 8 mL/min. The labeled product was
collected (Rt = 14 min) in a flask diluted with water (35 mL) and
loaded onto a C-18 cartridge (Water Sep-Pak, preactivated with
5 mL EtOH and 10 mL of water), and washed with additional
In vitro blood stability
The stability of compounds [11C]-3, [124I]-4, and [18F]-5 was
determined by the addition of 100, 25, and 50 mCi, respectively,
to 1 mL of human blood, plasma, and saline samples, followed
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J. Label Compd. Radiopharm 2009, 52 41–52

S. Dissoki et al.
by incubation at 371C for different time periods (0, 60, 90, and
120 min), under shaking conditions. All the blood samples were
centrifuged (eppendorf-centrifuge 5417C) at 3500 rpm for 5 min
to separate RBC from plasma. The radioactive material was
extracted by the addition of the solvent THF/CH3CN 30:70 (v/v)
to blood samples, vortexed for 0.5 min, and centrifuged at
10 500g for 5 min. The radioactivity was measured by a dose
calibrator (Capintec, CRC-35R), and then the upper organic
phase was loaded onto TLC plates (Partisils LK6DF Whatman,
reversed-phase silica gel). The total extracted radioactivity was
calculated by subtracting the non-extractable radioactivity from
the total one. TLC plates were run for 20 min with ammonium
formate 0.1 M (20%), CH3CN (75%), and THF (5%) as a mobile
phase. The TLC plates were exposed for 1 h to phosphor imager
plates (BAS-IP MS 2040 Fuji Photo Film Co., Ltd) for visualization
of radioactive bands. The plates were scanned with a BAS reader
3.1 version scanner, and analyzed with TINA 2.10 g software.
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Conclusion
We synthesized and radiolabeled with 11C, 18F, and 124I
pegylated anilinoquinazoline ML04 derivatives as candidates
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