Fluorine-18 labeling of ML04 - Presently the most promising irreversible inhibitor candidate for visualization of EGFR in cancer

Article abstract of DOI:10.1002/jlcr.1071

Overexpression of the EGFR has been linked to cell malignancy, metastasis and poor prognosis thus making it a target for several FDA approved drugs such as Gefitinib and Erlotinib. Unfortunately, these drugs have yielded suboptimal clinical results. In order to evaluate and monitor EGFR-targeted treatment response at the molecular level, several PET biomarkers have been developed. One of the lead irreversible inhibitors (1) has been labeled with carbon-11, however the short half-life of this radioisotope limited the time window for in vivo studies. Compound 1 was successfully labeled with fluorine-18 via a multi-step radiosynthesis with 14% decay-corrected overall radiochemical yield, 98% radiochemical purity, specific activity of 1800 Ci/mmol (n = 10) at end of bombardment, and a total radiosynthesis time of 4h including purification and formulation. [¹⁸F]-1 will allow for prolonged in vivo studies including Micro-PET analysis of EGFR tumor-bearing animal models. Copyright

Full text of DOI:10.1002/jlcr.1071

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 533–543.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1071
Research Article
Fluorine-18 labeling of ML04 – presently the most
promising irreversible inhibitor candidate for
visualization of EGFR in cancer
Samar Dissoki, Desideriu Laky and Eyal Mishani*
Department of Medical Biophysics and Nuclear Medicine, Hadassah Hebrew
University, Jerusalem 91120, Israel
Summary
Overexpression of the EGFR has been linked to cell malignancy, metastasis and poor
prognosis thus making it a target for several FDA approved drugs such as Gefitinib and
Erlotinib. Unfortunately, these drugs have yielded suboptimal clinical results. In order to
evaluate and monitor EGFR-targeted treatment response at the molecular level, several
PET biomarkers have been developed. One of the lead irreversible inhibitors ð1Þ has been
labeled with carbon-11, however the short half-life of this radioisotope limited the time
window for in vivo studies. Compound 1 was successfully labeled with fluorine-18 via a
multi-step radiosynthesis with 14% decay-corrected overall radiochemical yield, 98%
radiochemical purity, specific activity of 1800 Ci/mmol ðn ¼ 10Þ at end of bombardment,
and a total radiosynthesis time of 4 h including purification and formulation. ½18Fꢀ-1 will
allow for prolonged in vivo studies including Micro-PET analysis of EGFR tumor-bearing
animal models. Copyright # 2006 John Wiley & Sons, Ltd.
Received 14 February 2006; Accepted 10 March 2006
Key Words: EGFR; [18F]; PET; cancer
Introduction
Epidermal growth factor (EGF) and erb-2 (HER-2) kinase receptors are
among growth factor receptor kinases which play a major role in cancer in-
itiation, development and progression.1–4 Overexpression of the EGF receptor
*Correspondence to: Eyal Mishani, Hadassah University Hospital, PO Box 12000, Jerusalem 91120, Israel.
E-mail: mishani@md.huji.ac.il
Contract/grant sponsor: TK-Signal Ltd
Contract/grant sponsor: Rotem Industries Ltd
Copyright # 2006 John Wiley & Sons, Ltd.

534
S. DISSOKI ET AL.
F
Cl
Cl
HN
11CH3
N
H
N
N
H3C
O
N
Figure 1. Chemical structure of [11C]-ML04
(EGFR) or of one of its ligands has been linked to cell malignancy, metastasis, and
poor prognosis in patients.5 Thus, the EGFR has become an attractive target for
the design and development of compounds that can specifically bind the receptor,
and inhibit its tyrosine kinase activity and its signal transduction pathway in cancer
cells.6 The EGFR reversible inhibitor, Iressa1(ZD 1839, Gefitinib) was recently
approved by the FDA for treatment of non-small-cell-lung-carcinoma (NSCLC)
and prostate cancer.7 Gefitinib binds at the ATP site and inhibits the kinase
activity of EGFR and has been in clinical use since 2002. In addition, other anti-
EGFR targeted inhibitors, such as Tarceva# (OSI-774, Erlotinib) and the anti-
EGFR antibody Erbitux#, are presently undergoing clinical Phase 3 trials. Er-
lotinib and Gefitinib yield similar results in that they are both effective only in a
small percentage of patients in whom EGFR possesses activating mutations in the
kinase domain.8–10 Since accurate measurements of EGFR phosphorylation in the
human tumor are lacking, it is actually not possible to assess whether the poor
response to Gefitinib or Erlotinib is indeed due to a lack of the specific activating
mutations, the absence of a survival function of EGFR, or to insufficient long-term
occupancy of the receptor by reversible inhibitors. Consequently, 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 modality11–15 such
as positron emission tomography (PET). During the course of this research, a
potent ðIC50 ¼ 0:1 nMÞ and selective EGFR-TK irreversible inhibitor ML04 ð1Þ
was labeled with carbon-1111 (Figure 1), and its potential as PET biomarker was
investigated both in vitro and in vivo. Due to the short half-life of carbon-11 (t1/2
20.39 min), the in vivo studies in tumor-bearing rats with [11C]-ML04 were limited
to a short time window. However, since this inhibitor was presently proved to be
the most suitable candidate for visualization of EGFR in cancer, we were mo-
tivated to expand the study time window, and to label ML04 with a longer-lived
radioisotope, [18F] (t1/2 109.8 min), at the anilino moiety.
Results and discussion
The major disadvantage of labeling 1 with [11C] was the short half-life of the
isotope which limited the in vivo studies to rather short time periods. Since
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SUCCESSFUL [18F] LABELING OF EGFR IRREVERSIBLE INHIBITOR
535
[18F] has a longer half-life which enables for a longer follow-up period
of time following injection of the radiotracer, 1 was designated for labeling
with [18F]. With this in mind, two different precursors (2,3) were synthesized
for radiolabeling with [18F], requiring three additional steps after the labeling
step to yield the desired labeled compound. The radiosynthesis route
was based on a well-known nucleophilic substitution of the two leaving
groups: trimethylammuniumtriflate and a nitro group, attached at the anilino
moiety of compounds 2 and 3; respectively. The synthesis of precursor 2 is
outlined in Figure 2. 1,2-Dichloro-4,5-dinitro-benzene was reacted with hy-
drazine monohydrate and Raney-Nickel to yield compound 4 with 25% yield.
4-chloro-6-nitro-quinazoline in i-PrOH was added dropwise to compound
4 in the presence of triethylamine in i-PrOH and heated to 808C to
obtain compound 5 with 42% yield. Compound 5 was reacted with methyl-
trifluromethanesulfonate in the presence of 2,6-di-tert-butyl-4-methylpyridine
in dichloromethane/acetonitrile anhydrous for 6 h at RT to obtain compound
2 with 25% yield and 80–90% purity. Compound 2 was accompanied
by the by-product 6: It is well known that compounds containing the ammo-
nium quarternary salts such as compounds 2 and 6 are not stable and
sometimes their purification leads to decomposition.16 Although compounds 2
and 6 were purified by a short silica column, they could not be purified any
further by recrystallization. The synthesis of precursor 3 is outlined in
Figure 3. 4,5-Dichloro-2-nitro-aniline was refluxed with 4-chloro-6-nitro-
quinazoline and N,N-diisopropylethylamine in i-PrOH for 12 h to yield
compound 3 with a 20% yield and 97% purity. Attempts to radiolabel
H N
2
Cl
Cl
Cl
N
HN
H N
Cl
Cl
O N
2
2
O N
Cl
Cl
2
N
ii
i
+
O N
2
N
H N
2
O N
2
4
N
5
CF SO
3
3
CF SO
3
3
N
Cl
Cl
N
Cl
Cl
iii
HN
N
+
O N
O N
2
2
N
N
N
N
6
2
Figure 2. (i) H2NNH2.H2O, Ra-Ni, EtOH:H2O (9:1), 608C, 3h; (ii) i-PrOH,
Et3N, 808C, 5 h; (iii) CF3SO3CH3, 2,6-tert-butyl-4-methylpyridine, CH2Cl2/
CH3CN, RT, 6 h
Copyright # 2006 John Wiley & Sons, Ltd.
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536
S. DISSOKI ET AL.
O2N
HN
Cl
Cl
Cl
O2N
H2N
Cl
Cl
O2N
O2N
N
i
+
N
N
N
3
Figure 3. (i) i-PrOH, 908C, 4 h
Table 1. Attempts to radiolabel compounds 2; 3 with [18F] under different conditions
Temperature (8C)
Time (min)
Solvent
100, 120, 140
100, 120, 140
100, 120, 140
120
120
120
20
20
20
5, 10, 15
5, 10, 15
5, 10, 15
DMSO
CH3CN
DMF
DMSO
CH3CN
DMF
compounds 2; 3 with [18F] were performed in a commercial module under
different conditions such as time, solvent, and temperature (Table 1).
However, none of these experiments have been successful. In addition, reac-
tion of compound 6; which contains a tertiary amine, yielded similar
results and eliminated the possibility that the hydrogen of the secondary
amine in compounds 2 and 3 inactivated the fluoride by protonation,
and inhibited the transformation. Therefore, the most logical explanation
for the unsuccessful results is that the electron donating secondary
amine group in compounds 2 and 3 increased the electron density at the
aromatic ortho position attached to the two leaving groups of compounds 2; 3
and made the substitution unfeasible. Consequently, another radiosynthesis
route similar to the one that was developed for ML0117 and which was
recently used to label Gefitinib16 and other EGFR inhibitors,18 was per-
formed. However, using this process, we encountered several disadvantages,
including prolonged radiosynthesis time, radiation exposure and low yield due
to the half-life of [18F] (109.8 min) and the number of the radiochemical steps
that needed to be taken. To mitigate these problems, we designed and suc-
cessfully used a simple semi-automated radiosynthesis system that decreases
synthesis time and increases yield. The first two steps were performed in a
commercial module, and the last four with the homemade semi-automated
system. With this method, the exposure to radiation was decreased, and
the radiosynthesis time was cut down to a total of 4 h, including purification
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SUCCESSFUL [18F] LABELING OF EGFR IRREVERSIBLE INHIBITOR
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Cl
O2N
O2N
Cl
Cl
18F
Cl
Cl
18F
Cl
Cl
O2N
i
ii
N
iii
+
H2N
O2N
N
8
7
18F
HN
Cl
Cl
18F
Cl
Cl
18F
HN
Cl
Cl
HN
H
N
H2N
v
O2N
iv
N
N
N
N
O
vi
N
N
N
18F]-1
10
9
[
(ML04)
Figure 4. (i) K18F, kryptofix, DMF, 1158C, 20 min; (ii) H2NNH2.H2O, Ra-Ni,
EtOH:H2O (9:1), 588C, 7 min; (iii) i-PrOH, 100–1058C, 15 min; (iv)
H2NNH2.H2O, Ra-Ni, EtOH:H2O (9:1), 658C, 10 min; (v) bromo/chloro-
crotonylchloride, N,N-DIPEA, THF, 08C, 15 min; (vi) dimethylamine in THF
(2 M), 08C, 15 min
and formulation. The radiosynthesis of ½18Fꢀ-1 is shown in Figure 4.
1,2-Dichloro-4,5-dinitro-benzene was reacted with a solution of [18F] KF
and kryptofix in DMF at 1158C for 20 min to yield compound ½18Fꢀ-7 with
80% radiochemical yield. Reaction of the latter with hydrazine monohydrate
and Raney-Ni at 588C for 7 min yielded compound ½18Fꢀ-8 with 70% radi-
ochemical yield. The coupling of 4-chloro-6-nitro-quinazoline and ½18Fꢀ-8 was
performed in i-PrOH at 1008C to yield compound ½18Fꢀ-9 with 80% radio-
chemical yield. ½18Fꢀ-9 was reacted with hydrazine monohydrate and Raney-Ni
at 658C to obtain the reduced compound ½18Fꢀ-10 with 70% radiochemical
yield. Reaction of the latter with bromo/chlorocrotonylchloride and diisop-
ropylethylamine at 08C followed by reaction with dimethylamine provided
½18Fꢀ-1 ð½18Fꢀ-ML04Þ with 14% overall decay-corrected radiochemical yield,
98% radiochemical purity, specific activity of 1800 Ci/mmol ðn ¼ 10Þ and a
total radiosynthesis time of 4 h, including purification and formulation. For
comparison, 18F-Iressa was obtained after a three-step radiosynthesis (2 h),
with 60–70% overall decay-corrected radiochemical yield.16 However, it
should be noted that unlike compound 1 which is an irreversible EGFR in-
hibitor with a complex chemical structure,6 Iressa is a reversible inhibitor
which does not contain a complex reactive functional group at the six position
of the quinazoline ring that enables the in vivo covalent binding with the
receptor as does compound 1: Therefore, the fluorine-18 labeling of Iressa is
more straightforward. On the other hand, since Iressa is a reversible inhibitor,
it should compete with high concentrations of cellular ATP on receptor
Copyright # 2006 John Wiley & Sons, Ltd.
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538
S. DISSOKI ET AL.
binding and might yield a narrow time window for imaging immediately after
injection of the labeled biomarker.17
Experimental section
General
All chemicals were purchased from Sigma-Aldrich, Fisher Scientific, Merck or
J. T. Baker. Chemicals were used as supplied, excluding THF, which was
refluxed over sodium and benzophenone and freshly distilled prior to use.
Mass spectroscopy was performed in EI mode on a Thermo Quest-Finnigan
Trace MS-mass spectrometer at the Hadassah-Hebrew University Mass Spec-
troscopy Facility. 1H-NMR spectra were obtained on a Bruker AMX
300 MHz apparatus using the hydrogenated residue of the deuterated sol-
vents (DMSO-d6, d ¼ 2:5 ppm; CDCl3, d ¼ 7:25 ppm) and TMS as internal
1
standard for H-NMR. Elemental analysis was performed at the Hebrew
University Microanalysis Laboratory. Thin-layer chromatography (TLC) was
run on plates of silica gel 60F254 (Merck). The compounds were localized at
254 nm using a UV lamp. Reversed phase HPLC: analytical and semi-
preparative column C-18 mBondapak1 Waters, with mobile-phase system
composed of 45:55 (v/v) acetonitrile:acetate buffer 0.1 M pH 3.8 and 53:47 (v/
v) ammonium formate 0.1 M:acetonitrile, respectively. A Varian 9012Q pump,
a Varian 9050 variable wavelength detector operating at 254 nm, and a Bio-
scan Flow-Count radioactivity detector with a NaI crystal. Specific radioac-
tivities were determined by HPLC, using cold mass calibration lines.
Radiosyntheses using ½18Fꢀ were carried out on a [18F] module (Nuclear-
Interface, Munster, Germany). ½18Fꢀ was produced on a cyclotron IBA 18/9 by
irradiation of a 2 ml water target using a 18 MeV proton beam on 97%-
enriched [18O] water by the [18O(p,n)18F] nuclear reaction and was transferred
to the appropriate hot cell.
Chemistry
4,5-Dichloro-benzene-1,2-diamine (4)19. 1,2-Dichloro-4,5-dinitro-benzene (0.2
g, 0.84 mmol) dissolved in ethanol/water 9:1 (40 ml) was added dropwise to a
solution of ethanol/water 9:1 (15 ml), hydrazine monohydrate (4.2 mmol,
204.45 ml) and Raney Nickel solution (1 ml) at 608C. The reaction mixture was
stirred for an additional 1 h. The solution was cooled and filtered over Celite1,
and the filtrate was evaporated under reduced pressure. The residue was purified
on silica gel column, eluted with dichloromethane to yield 4,5-dichloro-benzene-
1,2-diamine 4 (0.05 g, 25%). TLC conditions: 1% MeOH in CH2Cl2, Rf ¼ 0:38:
HPLC conditions: C-18 column, 60% acetate buffer, pH ¼ 3:8; 40% acetonitrile,
1
flow=1 ml/min, Rt ¼ 7:2 min: H-NMR (DMSO): d 6.59 (s, 2H), 4.85 (s, 4H).
MS (m/z): 177.27 (MH)+. M.p.=1598C. (Lit., M.p.=1618C).19
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SUCCESSFUL [18F] LABELING OF EGFR IRREVERSIBLE INHIBITOR
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4,5-Dichloro-N-(6-nitro-quinazolin-4-yl)-benzene-1,2-diamine (5). 4-Chloro-
6-nitro-quinazoline11 (0.079 g, 0.37 mmol) dissolved in i-PrOH (60 ml) was
added dropwise to a solution of 4,5-dichloro-benzene-1,2-diamine (0.1 g,
0.56 mmol) and triethylamine (210 ml, 1.5 mmol) in i-PrOH (10 ml). The mix-
ture was heated to 808C for 5 h. The solution was cooled and evaporated, and
the product was purified on silica gel column, eluted with 2% MeOH in
dichloromethane to yield compound 5 (0.055 g, 42%). TLC conditions: 5%
MeOH in CH2Cl2, Rf ¼ 0:33: HPLC conditions: C-18 column, 60% acetate
buffer, pH ¼ 3:8; 40% acetonitrile, flow=1 ml/min, Rt ¼ 20 min:
1H-NMR(DMSO): d 8.9 (m, 1H), 8.84 (m, 1H), 8.63 (dm, 1H, J ¼ 9:3 Hz),
8.08 (dm, 1H, J ¼ 9 Hz), 7.65 (br s, 1H), 7.04 (br s, 1H). MS (m/z): 350.53
(MH)+. M.p.=192–1948C. Analytically calculated (C14H9Cl2N5O2.H2O): C,
45.65; H, 2.98; N, 19.0; Cl, 19.56. Found: C, 45.92; H, 2.80; N, 18.61; Cl, 20.9.
[4,5-Dichloro-2-(6-nitro-quinazolin-4-yl-amino)-phenyl]-trimethyl-ammonium-
triflate (2). 4,5-Dichloro-N-(6-nitro-quinazolin-4-yl)-benzene-1,2-diamine 5
(0.091 g, 0.26 mmol) was dissolved in CH2Cl2:CH3CN 5:1 (12 ml).
2,6-Di-tert-butyl-4-methylpyridine (0.053 g, 0.26 mmol) was added. After
5 min, methyltrifluromethanesulfonate (29.4 ml, 0.26 mmol) was added to the
basic solution. After 5 min, a dark-red color was produced. The reaction mix-
ture was stirred for 6 h and compound 2 was obtained with 65% conversion
accompanied by by-product 6: The crude product was purified on short silica
gel chromatography column, eluted with 2% MeOH in CH2Cl2 to yield com-
pound 2 (0.026 g, 25%). Compound 2 could not be purified further by re-
crystallization, and since the purity of the compound was only 85%, it was not
1
analyzed by elemental analysis. H-NMR(DMSO): d 8.93 (m, 1H), 8.62 (m,
1 H), 8.35 (m, 1H), 8.04 (m, 1H), 7.82 (dm, 1H), 7.68 (m, 1H), 7.59 (m, 1H),
3.68 (s, 9H). MS(m/z)=392.53 (MH)+.
{4,5-Dichloro-2-[methyl-(6-nitro-quinazolin-4-yl)-amino]-phenyl}-trimethyl-
ammonium-triflate (6). Compound 6 was simultaneously purified with 2; and
eluted with 5% MeOH in CH2Cl2 to yield 6 (0.005 g, 10%). 1H-NMR
(DMSO): d 8.6 (m, 1H), 8.34 (dm, 1H), 8.25 (m, 1H), 8.04 (m, 1H), 7.82 (m,
1H), 7.59 (m, 1H), 3.74 (s, 3H), 3.68 (s, 9H). MS(m/z)=406.4 (MH)+.
(4,5-Dichloro-2-nitro-phenyl)-(6-nitro-quinazolin-4-yl)-amine (3). 4-Chloro-
6-nitro-quinazoline (0.1 g, 0.47 mmol) was added to a solution of 4,5-
dichloro-2-nitro-aniline (0.197 g, 0.95 mmol) and N,N-diisopropylethylamine
(331 ml, 1.9 mmol) in i-PrOH (6 ml) and stirred at 908C for 12 h, yielding a
bright-brown precipitate. The solution was cooled, filtered, rinsed with i-PrOH
(10 ml), and dried in a vacuum oven to obtain 3 (0.036, 20%). TLC conditions:
2% MeOH in CH2Cl2, Rf ¼ 0:54: HPLC conditions: C-18 column, 60% ac-
etate buffer, pH ¼ 3:8; 40% acetonitrile, flow=1 ml/min, Rt ¼ 17:32 min:
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540
S. DISSOKI ET AL.
1H-NMR(DMSO): d 9.66 (s, 1H), 9.28 (m, 1H), 8.89 (m, 1H), 8.79 (m, 1H),
8.7 (dm, 1H, J ¼ 9), 8.45 (d, 1H, J ¼ 9:6 Hz), 8.06 (d, 1H, J ¼ 8:7 Hz).
MS(m/z): 381.2 (MH)+. M.p.=203–2058C. Analytically calculated
(C14H7Cl2N5O4.H2O): C, 42.20; H, 2.20; N, 17.58. Found: C, 42.33; H,
1.95; N, 18.2.
Radiochemistry
[18F]fluoride ion was produced using IBA cyclotron by irradiation of [18O]H2O
18
via the O(p,n)18F nuclear reaction. Syntheses of ½18Fꢀ-7 and ½18Fꢀ-8 were
performed in two commercial automated modules (Nuclear Interface,
Munster), and the last four steps with the homemade semi-automated sys-
¨
tem. The labeled compounds ½18Fꢀ-7-11; and ½18Fꢀ-1 were analyzed using
HPLC and compared to cold standards.6
1; 2-Dichloro-4-fluoro-5-nitro-benzene ([18F]-7). [18O]H2O/18Fꢁ (1930 mCi)
was trapped and then transferred through an ion exchange column (pre-
activated with 0.8 ml EtOH and 3 ml HPLC water) and by elution with 0.5 ml
potassium carbonate (2.5 mg/0.5 ml) to the reactor. A solution of 1 ml Kry-
ptofix1 222 (18 mg/1 ml CH3CN) was added. The water was removed by
azeotropic distillation with anhydrous acetonitrile at 958C under reduced
pressure for 3 min. 1,2-Dichloro-4,5-dinitro-benzene (10 mg) dissolved in
DMF (600 ml) was added. The reactor temperature was increased to 1158C,
and the reaction mixture was stirred for 20 min. The mixture was cooled to
308C, 13 ml of water was added to the reactor, and the mixture loaded on a C-
18 cartridge (Waters Sep-Pak, preactivated with 5 ml EtOH and 10 ml of sterile
water). Product ½18Fꢀ-7 was eluted with 2 ml EtOH to the collection vial, after a
total radiosynthesis time of 40 min and was obtained with 80% radiochemical
yield. The product was analyzed by a reverse-phase C-18 analytical column
and eluted with 55% acetate buffer 0.1 M/45% CH3CN, flow ¼ 1 ml=min;
Rt ¼ 14:3 min:
4; 5-Dichloro-2-fluoro-aniline ([18F]-8). Hydrazine monohydrate (200 ml) was
added to a reactor containing 200 ml of EtOH:H2O 9:1 and Ra-Ni (400 ml), and
½18Fꢀ-7 in EtOH/H2O was added drop-wise to the reactor. The synthesis was
proceeded at 608C for 7 min to yield ½18Fꢀ-8 with 70% radiochemical yield. The
product was diluted with 10 ml water, passed through a C-18 cartridge, and
washed with water (5 ml); the column was dried under argon for 5 min. The
product was eluted with i-PrOH (2.5 ml), and analyzed by a reverse-phase
C-18 analytical column, and eluted with 55% acetate buffer 0.1 M/45%
CH3CN, flow ¼ 1 ml=min; Rt ¼ 11:66 min:
Copyright # 2006 John Wiley & Sons, Ltd.
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SUCCESSFUL [18F] LABELING OF EGFR IRREVERSIBLE INHIBITOR
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(4,5-Dichloro-2-fluoro-phenyl)-(6-nitro-quinazolin-4-yl)-amine ([18F]-9). 4-
Chloro-6-nitro-quinazoline (20 mg) dissolved in i-PrOH (0.5 ml) was added
to a solution of ½18Fꢀ-8 in i-PrOH (2.5 ml). The reaction mixture was refluxed at
100–105 8C for 5 min and a second batch (20 mg) was added. The reaction
mixture was refluxed for an additional 15 min to obtain ½18Fꢀ-9 with 80%
radiochemical yield. The product was analyzed by a reverse-phase analytical
column, and eluted with 55% acetate buffer 0.1 M/45% acetonitrile, flow ¼
1 ml=min; Rt ¼ 20:92 min:
N4-(4,5-Dichloro-2-fluoro-phenyl)-quinazoline-4,6-diamine ([18F]-10). The vial
containing ½18Fꢀ-9 was transformed into another heating bath (658C),
EtOH:H2O 9:1 (250 ml), hydrazine monohydrate (450 ml) and Ra-Ni (400 ml)
were added. The reaction mixture was stirred and heated to 658C for 10min to
obtain ½18Fꢀ-10 with 70% radiochemical yield. The solution was cooled, diluted
with water (10 ml) and loaded onto a C-18 Sep-pak; the column was dried under
argon for 10min for the next step. The product was analyzed by a reverse-phase
analytical column, and eluted with 55% acetate buffer 0.1 M/45% acetonitrile,
flow ¼ 1 ml=min; Rt ¼ 9:73 min:
N-{4-[(4,5-dichloro-2-fluorophenyl)amino]quinazolin-6-yl}-dimethylamine-
butynamide ([18F]-1). ½18Fꢀ-10 was eluted with dry THF (2.5 ml) to a conical
vial at 08C. Two hundred and fifty microliters of N,N-diisopropylethylamine
in THF (20 ml/ml), and 0.8 ml of Br/Cl-crotonylchloride20 in THF (100 mg/
1 ml) were added. The reaction mixture was stirred for 20 min at 08C, and used
for the next step without any further treatment. The product was analyzed by
a C-18 analytical column under the same conditions, Rt ¼ 21:5 min
(first peak), Rt ¼ 23:8 min (second peak). Dimethylamine (2.0 M) in
THF (1 ml) was added to the solution at 08C and the reaction proceeded for
15 min. The solution was evaporated under argon to a volume of 700 ml,
then a mixture of CH3CN:H2O (1:1) was added. ½18Fꢀ-1 was analyzed
by a reverse-phase C-18 column, Rt ¼ 11:3 min; and purified using an
HPLC reversed-phase C-18 preparative column to yield ½18Fꢀ-ML04 with an
average of 14% radiochemical yield, specific activity of 1800 Ci/mmol, and
98% radiochemical purity ðn ¼ 10Þ: HPLC conditions: C-18 preparative col-
umn, 53% ammonium formate 0.1 M/47% acetonitrile, flow ¼ 5 ml=min;
Rt ¼ 35 min:
Conclusion
ML04 was successfully labeled with ½18Fꢀ at the anilino moiety, by six
radiochemical steps using two commercial modules and a homemade semi-
automated system. [18F]-ML04 was obtained after 4 h synthesis time, including
purification and formulation with
a
14% decay-corrected overall
radiochemical yield, specific activity of 1800 Ci/mmol ðn ¼ 10Þ at EOB, and
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49: 533–543
DOI: 10.1002/jlcr

542
S. DISSOKI ET AL.
radiochemical purity of 98%. [18F]-ML04, will allow for prolonged in vivo
studies including Micro-PET analysis of EGFR tumor-bearing animal models.
Acknowledgements
Support was provided by TK-Signal Ltd, Jerusalem, Israel, and by Rotem
Industries Ltd, Beer-Sheva, Israel.
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