Fluorine-18 labeling of 6,7-disubstituted anilinoquinazoline derivatives for positron emission tomography (PET) imaging of tyrosine kinase receptors: Synthesis of18F-Iressa and related molecular probes

Article abstract of DOI:10.1002/jlcr.998

Inhibitors of tyrosine kinase enzymatic activity represent a promising new class of antineoplastic agents. Although clinical studies performed over the last decade give more insight on the potential therapeutic applications of such drugs, identification of the individual patients who might benefit from them remains a major challenge. We have developed a synthetic strategy for the production of a wide variety of radiolabeled 6,7-disubstituted 4-anilinoquinazolines suitable for noninvasive imaging of tyrosine kinase receptors to predict therapy effectiveness. Three new F-18 labeled radiopharmaceuticals based on the therapeutic agents Tarceva, Iressa, and ZD6474 were synthesized. Decay-corrected yields varied between 25 and 40% for a total synthesis time of 120 min, thus providing F-18 labeled tyrosine kinase inhibitors in quantities and times practical for use as PET radiopharmaceuticals. Copyright

Full text of DOI:10.1002/jlcr.998

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 829–843.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.998
Research Article
Fluorine-18 labeling of 6,7-disubstituted
anilinoquinazoline derivatives for positron emission
tomography (PET) imaging of tyrosine kinase
receptors: synthesis of F-Iressa and related
18
molecular probes
Yann Seimbille*, Michael E. Phelps, Johannes Czernin and Daniel H. S.
Silverman
Ahmanson Biological Imaging Division, Department of Molecular and Medical
Pharmacology, University of California, Los Angeles, CA 90095-6942, USA
Summary
Inhibitors of tyrosine kinase enzymatic activity represent a promising new class of
antineoplastic agents. Although clinical studies performed over the last decade give
more insight on the potential therapeutic applications of such drugs, identification of
the individual patients who might benefit from them remains a major challenge.
We have developed a synthetic strategy for the production of a wide variety
of radiolabeled 6,7-disubstituted 4-anilinoquinazolines suitable for noninvasive
imaging of tyrosine kinase receptors to predict therapy effectiveness. Three new
F-18 labeled radiopharmaceuticals based on the therapeutic agents Tarceva, Iressa,
and ZD6474 were synthesized. Decay-corrected yields varied between 25 and 40% for
a total synthesis time of 120 min, thus providing F-18 labeled tyrosine kinase
inhibitors in quantities and times practical for use as PET radiopharmaceuticals.
Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: EGFR; tyrosine kinase inhibitors; fluorine-18; positron emission
tomography
*Correspondence to: Yann Seimbille, Ahmanson Biological Imaging Division, UCLA School of Medicine,
Department of Molecular and Medical Pharmacology, University of California, 10833 Le Conte Avenue,
CHS AR-264, Los Angeles, CA 90095-6942, USA. E-mail: yseimbille@mednet.ucla.edu
Contract/grant sponsor: California Breast Cancer Research Program; contract/grant numbers: 7KB-0155,
DOE DE-FC03-87ER60615
Copyright # 2005 John Wiley & Sons, Ltd.
Received 23 March 2005
Revised 5 May 2005
Accepted 6 July 2005

830
Y. SEIMBILLE ET AL.
Introduction
The epidermal growth factor receptor (EGFR) signaling pathway contributes
to a number of processes important to cancer development and progression.1,2
Over the last decade, selective small-molecule inhibitors of EGFR tyrosine
kinase enzymatic activity have been focused upon as potential anti-cancer
agents and as experimental tools for understanding the physiological roles
of EGFR signaling.3 Several 6,7-disubstituted 4-anilinoquinazolines have
demonstrated promising antitumor activity in vitro and in vivo and
are currently in clinical trials (Figure 1).4,5 One of them, Iressa has recently
been approved for treatment of advanced lung cancer.6 Introduction of
C-7 basic side chains to 4-anilinoquinazolines induces additional activity
against vascular endothelial growth factor receptor (VEGFR), known to
play a key role in tumor angiogenesis.7 However, the clinical expectations
have not been fulfilled yet, and one needs to better identify the subset of
patients that will benefit from EGFR-inhibitor mediated therapy. Clinical
deployment of these compounds would also be greatly facilitated by
non-invasive imaging techniques with which to measure drug concentrations
in the tumor tissue and monitor inhibition of EGFR and VEGFR kinase
activity.2,6
A substantial number of ATP competitive inhibitors of the EGFR kinase
have already been radiolabeled and evaluated as potential imaging agents.8–16
However, so far significant EGFR specific tumor uptake has not been
convincingly demonstrated in vivo. Furthermore, those approaches used
EGFR kinase inhibitors not studied in clinical trials, and it is not clear whether
those imaging agents can actually be used to assess the biodistribution of
clinically used EGFR inhibitors. Our strategy was therefore to develop
radiopharmaceuticals with structures identical or very closely related to
therapeutic agents that have proven to be clinically successful.
In the present paper, we report a versatile and parallel synthetic route to
prepare the different key intermediate 4-chloroquinazolines 8 (Scheme 1).
Specific substituents were introduced at the 6-, or 7-, or both positions; well-
known for their interactions with key amino acids of EGFR and/or VEGFR.
Br
F
F
4'
3'
HN
N
HN
O
HN
Cl
4
O
O
N
O
O
6
7
O
O
N
O
O
N
N
N
N
N
Iressa
Tarceva
ZD6474
Figure 1. Promising inhibitors of EGFR and VEGFR activity
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF 18F-IRESSA AND RELATED MOLECULAR PROBES
831
NO2
COOMe
COOMe
From 5 to 9:
TFA / HNO3
a: R = Et; R1 = R2 = CH3OCH2CH2
MeO
MeO
O
O
b: R = Me; R1 = CH3; R1 =
O(CH2)3Cl
O(CH2)3Cl
N
3
4
c: R = Me; R1 = CH3; R2 =
N
NH
NO2
NH2
COOR
COOR
COOR
TFA / HNO3
H2, Pd/C
R2O
R2O
R2O
OR1
OR1
OR1
1: R = Et ; R1 = R1 = CH3OCH2CH2
5
6
2: R = Me ; R1 = CH3 ; R2 =
N
HCONH2
F
F
Cl
Cl
O
N
HN
R1O
R2O
R1O
R2O
R1O
Cl
H2N
N
NH
Cl(CO)2Cl
N
IPA or DMF
N
R2O
N
9
8
7
Scheme 1. Synthesis of 6,7-disubstituted anilinoquinazoline derivatives
The synthetic strategy proposed here can be used to produce a library of 6,7-
disubstituted 4-anilinoquinazolines, and subsequently radiolabeled analogs for
imaging purposes. Three non-radioactive tyrosine kinase inhibitors (TKIs)
were synthesized by condensation of 3-chloro-4-fluoroaniline with the 4-
chloroquinazoline intermediates with over 50% yield. We have also
investigated the fluorine-18 labeling of those TKIs by coupling of the 4-
chloroquinazolines 8 with 3-chloro-4-[18F]fluoroaniline ð12Þ.
Results and discussion
Chemistry
Anilinoquinazolines 9 were prepared by one main route consisting of the
following steps: (1) introduction of the substituents, different from the
methoxy group, at the C-6 and/or C-7 positions of the quinazoline nucleus;
(2) nitration and subsequent reduction of the nitro group to provide 2-
aminobenzoate derivatives; (3) formation of the 4-quinazolones; (4) conver-
Copyright # 2005 John Wiley & Sons, Ltd.
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832
Y. SEIMBILLE ET AL.
sion of the quinazolones into 4-chloroquinazolines; and (5) attachment of the
aniline ring (Scheme 1).
Alkylation at both 3- and 4-position by treatment of ethyl 3,4-dihydrox-
ybenzoate with 2-bromoethyl methyl ether in presence of base, followed by
nitration at the 2-position of ethyl 3,4-bis(2-methoxyethoxy)benzoate ð1Þ with
nitric acid in TFA gave the key intermediate 5a in 60% yield from ethyl 3,4-
dihydroxybenzoate.17 The disubstituted derivative 5a was subsequently used
to obtain a quinazoline structure similar to Tarceva, possessing similar
substituents at both 6- and 7-positions.
Introduction of the 3-propoxymorpholino group found in Iressa was
realized by treatment of methyl 3-hydroxy-4-methoxybenzoate with 1-bromo-
3-chloropropane in presence of potassium carbonate.18 Aromatic nitration of
3, and nucleophilic displacement of the aliphatic chlorine of 4 by heating in an
excess of morpholine led to the expected nitrobenzoate derivative 5b in 37%
yield from methyl 3-hydroxy-4-methoxybenzoate.
Reaction of methyl vanillate with N-BOC-4-piperidinemethanol under
Mitsunobu conditions using diethyl azodicarboxylate afforded methyl 3-
methoxy-4-(1-tert-butyloxycarbonylpiperidin-4-ylmethoxy)benzoate. The t-
BOC protecting group was subsequently removed by treatment with
concentrated HCl in 1,4-dioxane, and the piperidine nitrogen was methylated
under Eschweiler–Clarke (HCOOH, HCHO, reflux) to yield 2, containing the
C-7 basic side chain of ZD6474.19 Treatment of 2 with fuming HNO3 in TFA
provided methyl 5-methoxy-4-(1-methylpiperidin-4-ylmethoxy)-2-nitrobenzo-
ate ð5cÞ in 56% yield from methyl vanillate.
Reduction of the nitro group of the intermediates 5 by catalytic
hydrogenation and subsequent Niementowski condensation gave the corre-
sponding quinazolones 7. Treatment of the quinazolones with oxalyl chloride
yielded quantitatively the 4-chloroquinazolines 8. The preparation of the non-
radioactive tyrosine kinase inhibitors 9 was achieved by condensation of
3-chloro-4-fluoroaniline with the substituted 4-chloroquinazolines 8 in
2-propanol or DMF with over 50% yield.
Radiochemistry
The radiolabeled EGFR inhibitors were prepared starting with the standard
kryptofix-K2CO3-mediated nucleophilic 18F exchange reaction with a
trimethylammonium triflate precursor 10 (Scheme 2). Introduction of the
fluorine-18 using a no-carrier-added nucleophilic substitution with K[18F]F-
K222 (K222
:
Kryptofix [2.2.2]; 4,7,13,16,21,24-hexaoxa-1,10-diazabicy-
clo[8.8.8]hexacosane) was initially conducted in N,N-dimethyl sulfoxide
(sealed V-vial) at 1308C for 10 min, leading to a rapid degradation of the
precursor and low radiochemical yields (Table 1). Therefore, we investigated
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF 18F-IRESSA AND RELATED MOLECULAR PROBES
833
whether we could improve the nucleophilic incorporation of [18F]fluoride into
the activated trimethylammonium triflate salt by changing the reaction
conditions (temperature, time, and solvent). The reaction was then performed
in either DMSO or CH3CN at 1108C, but no significant improvement was
observed. Although trimethylammonium triflate salts have been widely used
for fluoride ion nucleophilic aromatic substitution reactions, they are known
to be thermally unstable. Therefore, we sought to accomplish the first
radiochemical step at room temperature, and yields, with respect to initial
[18F]fluoride, were approximately 75% (decay corrected) when the reaction
occurred in acetonitrile for 25 min vs 20% when the same reaction mixture was
heated at 1108C for 15 min.
Then, reduction of the nitro with sodium borohydride in presence
of palladium on activated carbon gave 3-chloro-4-[18F]fluoroaniline ð12Þ
(Table 1). Typically, starting from 3.4 to 6.1 GBq (92–165 mCi) aliquot of a
cyclotron-produced [18F]Fꢀ batch, 1.4–2.2 GBq (38–60 mCi) of 3-chloro-4-
[18F]fluoroaniline could be obtained in 70–80 min.
Subsequent coupling of 3-chloro-4-[18F]fluoroaniline ð12Þ with the 4-
chloroquinazoline intermediates 8 in 2-propanol afforded, after HPLC
purification, the F-18 labeled 4-anilinoquinazolines in 25–40% decay corrected
+NMe3 -OTf
F18
Cl
F18
Cl
K[18F]F / K2CO3-K222
NaBH4 - Pd/C
O2N
Cl
O2N
H2N
10
11
12
Scheme 2. Preparation of 3-chloro-4-[18F]fluoroaniline ð12Þ
Table 1. Production of 3-chloro-4-[18F]fluoroaniline ð12Þ
Solventa
Temperature
Timec
RCYd
Step 1
A
A
A
1308C
1108C
1108C
10
15
20
33.3–34%
20.8–44.4%
39.1–46.2%
B
B
B
B
1108C
RTb
RT
15
15
20
25
20%
46.1–49.3%
52.8–58.9%
71.1–75.8%
RT
Step 2
MeOH
RT
5
95.2–97.7%
a Solvent A: anhydrous N,N-dimethyl sulfoxide; solvent B: anhydrous acetonitrile.
b RT ¼ room temperature.
c Time is expressed in minutes.
d RCY ¼ radiochemical yield (decay corrected).
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834
Y. SEIMBILLE ET AL.
F18
Cl
Cl
N
F18
R1O
HN
N
N
IPA
R1O
R2O
+
R2O
N
H2N
Cl
8a: R1 = R2 = CH3OCH2CH2
12
O
[18F]-9a: R1 = R2 = CH3OCH2CH2
8b: R2 = CH3 ; R1
=
=
N
O
[18F]-9b: R2 = CH3 ; R1
=
N
8c: R1 = CH3 ; R2
N
[18F]-9c: R1 = CH3 ; R2
=
N
Scheme 3. Synthesis of F-18 labeled anilinoquinazolines ([18F]-9a–c)
radiochemical yields (Scheme 3). The average total synthesis time was about
2 h. Identities of the new radiopharmaceuticals were confirmed by comparing
their HPLC mobility with those of the non-radioactive analogues. Specific
activity, determined by using on-line measurements of radioactivity and UV
absorption, was >222 GBq/mmol (>6000 Ci/mmol).
Experimental
General
All reagents, including anhydrous solvents, were purchased from Aldrich,
Sigma and Acros, and were used without further purification. Analytical thin
layer chromatography (TLC) was performed on Sigma-Aldrich silica gel pre-
coated plastic sheets with fluorescent indicator (UV 254). Visualization was
achieved with short-wave ultraviolet light. Column chromatography was
performed using silica gel (60–200 mesh). High performance liquid chromato-
graphy (HPLC) was carried out on an Agilent 1100 Series system. HPLC
eluates were monitored for their UV absorbency at 254 nm, and their
radioactivity content by connecting the outlet of the UV-photometer to a NaI
detector. The recorded data were processed by an Agilent ChemStation
software system. 18F-activity was also measured with an ion chamber Capintec
CRC11. The HPLC column used for the characterization of the product
against standards and for the purification of radioactive compounds was a
Phenomenex Luna-C18 (5 mm, 250 mm ꢁ 10 mm) column, operated at a flow
1
rate of 2 ml/min. H NMR spectrum was taken in deuterated solvents on a
Bruker AM360/Wb spectrometer (at 360 MHz) using Me4Si as an internal
standard, and selected proton resonances are reported. Chemical shifts are
expressed in ppm (d) relative to the standard and coupling constants (J) in Hz.
Mass spectrum was obtained on MALDI-TOF instrument (ABI DE STR) and
EI instrument (Micromass, Waters Autospec).
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 829–843

SYNTHESIS OF 18F-IRESSA AND RELATED MOLECULAR PROBES
835
Chemistry
Synthesis of ethyl 3,4-bis(2-methoxyethoxy)benzoate ð1Þ. To ethyl 3,4-dihy-
droxybenzoate (2.6 g, 14.3 mmol), potassium carbonate (4.35 g, 31.5 mmol)
and tetrabutylammonium iodide (54 mg, 0.14 mmol) in degassed acetone
(30 ml) was added 2-bromoethyl methyl ether (5 g, 36 mmol). The mixture was
stirred under argon at reflux for 72 h. Ether (45 ml) was added to the mixture
and after stirring 30 min at room temperature, the precipitate was removed by
filtration. The filtrate was concentrated in vacuo and the residue was triturated
1
with hexane to afford 1, as a white solid (2.9 g, 68%). H NMR (360 MHz,
CDCl3): d : 7:67 (dd, J ¼ 2:1 and 8.6 Hz, 1H), 7.58 (d, J ¼ 2:1 Hz, 1H), 6.91
(d, J ¼ 8:3 Hz, 1H), 4.34 (q, J ¼ 7:2 Hz, 2H), 4.20 (m, 4H), 3.80 (m, 4H), 3.46
(s, 6H), 1.38 (t, J ¼ 7:2 Hz, 3H). HRMS (EI) m/z: calculated for C15H22O6,
298.1416; found, 298.1422.
Synthesis of methyl 3-methoxy-4-(1-methylpiperidin-4-ylmethoxy)benzoate
ð2Þ. Triphenylphosphine (3.4 g, 13 mmol) was added under argon to a
suspension of methyl vanillate (1.82 g, 10 mmol) in CH2Cl2 (40 ml), followed
by the addition of N-BOC-piperidinemethanol (2.58 g, 12 mmol) and by a
solution of diethyl azodicarboxylate (40% in toluene, 13 mmol) in CH2Cl2
(10 ml). After the solution was stirred for 1 h at ambient temperature, the
reaction mixture was poured onto a column of silica and was eluted with
hexane/EtOAc (from 10/0 to 3/1) to afford methyl 3-methoxy-4-(1-tert-
butyloxycarbonylpiperidin-4-ylmethoxy)benzoate (3.49 g, 92%). 1H NMR
(360 MHz, CDCl3): d : 7:65 (dd, J ¼ 1:7 and 8.6 Hz, 1H), 7.55 (s, 1H), 6.85
(d, J ¼ 8:3 Hz, 1H), 4.30–4.05 (m, 2H), 3.91 (s, 3H), 3.89 (s, 3H), 2.75 (m, 2H),
2.15–2.00 (m, 2H), 1.87 (d, J ¼ 12:6 Hz, 2H), 1.46 (s, 9H), 1.26 (m, 3H).
HRMS (EI) m/z: calculated for C20H29NO6, 379.1995; found, 379.2002.
Methyl 3-methoxy-4-(1-tert-butyloxycarbonylpiperidin-4-ylmethoxy)benzoate
(3 g, 7.9 mmol) was dissolved in 1,4-dioxane (5 ml) and treated with
concentrated HCl (1 ml) for 1 h at room temperature. The volatiles were
evaporated under vacuum, and the crude material was subsequently dissolved
in formic acid (3.5 ml). Formaldehyde (12 M, 37% in water, 3.5 ml) was added
and the mixture was stirred overnight at 958C. The solvent was removed under
reduced pressure and the residue was dissolved in water (30 ml). The pH was
adjusted to 11 with an aqueous solution of sodium hydroxide (2 M). The
mixture was diluted with ethyl acetate and the organic layer was separated.
The aqueous layer was further extracted with ethyl acetate, and the organic
layers were combined. The organic layers were washed with water, brine, dried
over Na2SO4, and concentrated under reduced pressure to give the title
1
compound (1.85 g, 80%). H NMR (360 MHz, DMSO-d6): d : 7:56 (dd, J ¼
2:1 and 8.3 Hz, 1H), 7.44 (d, J ¼ 2:1 Hz, 1H), 7.07 (d, J ¼ 8:3 Hz, 1H), 3.88 (d,
J ¼ 5:8 Hz, 2H), 3.81 (s, 3H), 3.80 (s, 3H), 2.76 (d, J ¼ 11:5 Hz, 2H), 2.15
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Y. SEIMBILLE ET AL.
(s, 3H), 1.85 (t, J ¼ 11:5 Hz, 2H), 1.72 (m, 3H), 1.29 (m, 2H). HRMS (EI) m/z:
calculated for C16H23NO4, 293.1627; found, 293.2629.
Synthesis of methyl 3-(3-chloropropoxy)-4-methoxybenzoate ð3Þ. To methyl 3-
hydroxy-4-methoxybenzoate (3.75 g, 20.6 mmol) and potassium carbonate
(5 g, 36.2 mmol) in degassed acetone (25 ml) was added 1-bromo-3-chlor-
opropane (8 g, 50.8 mmol). The mixture was stirred under Argon at reflux for
24 h. The precipitate was removed by filtration and washed with ethyl acetate.
The volatiles were removed under reduced pressure. The resulting solid was
triturated in hexane, filtered, and dried under vacuum to give 3 (4.08 g, 76%).
1H NMR (360 MHz, CDCl3): d : 7:69 (dd, J ¼ 2:1 and 8.3 Hz, 1H), 7.57 (d,
J ¼ 2:1 Hz, 1H), 6.90 (d, J ¼ 8:6 Hz, 1H), 4.22 (t, J ¼ 6:1 Hz, 2H), 3.92 (s,
3H), 3.89 (s, 3H), 3.78 (t, J ¼ 6:1 Hz, 2H), 2.31 (q, J ¼ 6:1 Hz, 2H). HRMS
(EI) m/z: calculated for C12H15O4Cl, 258.0659; found, 258.0655.
Synthesis of methyl 5-(3-chloropropoxy)-4-methoxy-2-nitrobenzoate ð4Þ. A
solution of 3 (2 g, 7.7 mmol) in methylene chloride (8 ml) was cooled to
0–58C. TFA (4 ml) was added followed by addition in portions over 15 min of
a solution of fuming 24 M nitric acid (0.8 ml) in methylene chloride (1.6 ml).
After completion of the addition, the solution was allowed to warm up and
stirred at ambient temperature for 2 h. The volatiles were removed by
evaporation, and the residue was purified by column chromatography eluting
with CH2Cl2/EtOAc (from 10/0 to 9/1). Evaporation of the fractions
containing the expected product gave 1.92 g of 4 (82%), as an orange oil.
1H NMR (360 MHz, CDCl3): d : 7:45 (s, 1H), 7,11 (s, 1H), 4.26 (t, J ¼ 6:1 Hz,
2H), 3.95 (s, 3H), 3.91 (s, 3H), 3.77 (t, J ¼ 6:1 Hz, 2H), 2.33 (q, J ¼ 6:1 Hz,
2H). HRMS (EI) m/z: calculated for C12H14NO6Cl, 303.0510; found,
303.0502.
A similar procedure was used to prepare ethyl 4,5-bis(2-methoxyethoxy)-2-
nitrobenzoate ð5aÞ and methyl 5-methoxy-4-(1-methylpiperidin-4-ylmethoxy)-
2-nitrobenzoate ð5cÞ. 5a: 1H NMR (360 MHz, CDCl3): d : 7:49 (s, 1H), 7.10 (s,
1H), 4.35 (q, J ¼ 7:2 Hz, 2H), 4.23 (m, 4H), 3.78 (m, 4H), 3.44 (s, 6H), 1.33 (t,
J ¼ 7:2 Hz, 3H). HRMS (EI) m/z: calculated for C15H21NO8, 343.1267; found,
1
343.1270. 5c: H NMR (360 MHz, DMSO-d6): d : 7:67 (s, 1H), 7.35 (s, 1H),
4.04 (d, J ¼ 6:1 Hz, 2H), 3.93 (s, 3H), 3.83 (s, 3H), 3.47 (m, 2H), 2.97 (m, 2H),
2.77 (s, 3H), 2.10–1.80 (m, 3H), 1.48 (m, 2H). HRMS (EI) m/z: calculated for
C16H22N2O6, 338.1478; found, 338.1475.
Synthesis of methyl 4-methoxy-5-(3-morpholinopropoxy)-2-nitrobenzoate
ð5bÞ. Morpholine (5 ml) was added to a suspension of 4 (1.83 g, 6 mmol)
and the solution was heated at 1008C for 1 h. The residue was diluted with
ethyl acetate and poured into ice cold water. The organic layer was washed
with a 5% aqueous solution of NaHCO3, twice with H2O, dried over Na2SO4,
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF 18F-IRESSA AND RELATED MOLECULAR PROBES
837
and concentrated under reduced pressure to give the title compound (1.28 g,
1
60%). H NMR (360 MHz, CDCl3): d : 7:45 (s, 1H), 7.10 (s, 1H), 4.18 (t,
J ¼ 6:8 Hz, 2H), 3.94 (s, 3H), 3.90 (s, 3H), 3.70 (m, 4H), 2.51 (t, J ¼ 6:8 Hz,
2H), 2.45 (m, 4H), 2.05 (q, J ¼ 6:8 Hz, 2H). HRMS (EI) m/z: calculated for
C16H22N2O7, 354.1427; found, 354.1417.
Synthesis of ethyl 2-amino-4,5-bis(2-methoxyethoxy)benzoate ð6aÞ. A suspen-
sion of 5a (2.06 g, 6 mmol) in methanol (15 ml) containing 10% palladium on
activated carbon (300 mg) was hydrogenated until uptake of hydrogen ceased.
The mixture was filtered and the volatiles were removed by evaporation. The
residue was dissolved in ether and 2 M hydrogen chloride in ether (5 ml) was
added. The solid was collected by filtration and dried under vacuum to give 6a
(1.76 g, 94%). 1H NMR (360 MHz, CDCl3): d : 7:42 (s, 1H), 6.12 (s, 1H), 5.61
(brs, 2H), 4.27 (q, J ¼ 7:2 Hz, 2H), 4.07 (m, 4H), 3.71 (m, 4H), 3.43 (s, 6H),
1.34 (t, J ¼ 7:2 Hz, 3H). HRMS (EI) m/z: calculated for C15H23NO6,
313.1525; found, 313.1531.
A similar procedure was used to prepare methyl 2-amino-4-methoxy-5-(3-
morpholinopropoxy)benzoate ð6bÞ and methyl 2-amino-5-methoxy-4-
(1-methylpiperidin-4-ylmethoxy)benzoate ð6cÞ. 6b: 1H NMR (360 MHz,
CDCl3): d : 7:34 (s, 1H), 6.14 (s, 1H), 5.61 (brs, 2H), 4.00 (t, J ¼ 6:5 Hz,
2H), 3.84 (s, 3H), 3.83 (s, 3H), 3.73 (m, 4H), 2.54 (t, J ¼ 7:2 Hz, 2H), 2.48 (m,
4H), 1.99 (q, J ¼ 6:8 Hz, 2H). HRMS (EI) m/z: calculated for C16H24N2O5,
1
324.1685; found, 324.1675. 6c: H NMR (360 MHz, DMSO-d6): d : 7:20 (s,
1H), 6.62 (s, 1H), 3.82 (d, J ¼ 6:5 Hz, 2H), 3.76 (s, 3H), 3.69 (s, 3H), 3.42 (m,
2H), 2.96 (m, 2H), 2.70 (s, 3H), 2.10–1.90 (m, 3H), 1.61 (m, 2H). HRMS (EI)
m/z: calculated for C16H24N2O4, 308.1736; found, 308.1729.
Synthesis of 6,7-bis(2-methoxyethoxy)-3,4-dihydroquinazolin-4-one ð7aÞ. 6a
(1.3 g, 4.1 mmol) and ammonium formate (260 mg, 4.1 mmol) were dissolved
in formamide (2.2 ml) and the stirred mixture was heated to 1608C under
an atmosphere of Argon for 3 h. After cooling, water (7 ml) was added and the
precipitate was recovered by filtration, washed with cold water and dried. The
filtrate was extracted five times with CHCl3, and the pooled organic extracts
were washed with brine, dried over Na2SO4, and concentrated. The residue
and crude quinazolone precipitate were combined, triturated in hot CH3CN
(9 ml) for 30 min, cooled to 208C, and treated with ether (9 ml). After cooling
to 48C, 7a was recovered by filtration as a white solid and dried (753 mg, 62%).
1H NMR (360 MHz, CD3OD): d : 7:98 (s, 1H), 7.61 (s, 1H), 7.15 (s, 1H), 4.28
(m, 4H), 3.85 (m, 4H), 3.48 (s, 6H). HRMS (EI) m/z: calculated for
C14H18N2O5, 294.1216; found, 294.1213.
Synthesis of 7-methoxy-6-(3-morpholinopropoxy)-3,4-dihydroquinazolin-4-one
ð7bÞ. A solution of 6b (650 mg, 2 mmol) in 2-methoxyethanol (8 ml) containing
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Y. SEIMBILLE ET AL.
formamidine acetate (208 mg, 2 mmol) was heated at 1158C for 2 h.
Formamidine acetate (416 mg, 4 mmol) was added in portions every 30 min
during 4 h. Heating was prolonged for 30 min after the last addition. After
cooling, the volatiles were removed under vacuum and the residue applied to
flash chromatography (CH2Cl2/MeOH from 10/0 to 9/1) to give 7b (498 mg,
1
78%). H NMR (360 MHz, DMSO-d6): d : 7:99 (s, 1H), 7.43 (s, 1H), 7.12 (s,
1H), 4.10 (t, J ¼ 6:5 Hz, 2H), 3.90 (s, 3H), 3.83 (s, 3H), 3.57 (m, 4H), 2.42 (t,
J ¼ 6:8 Hz, 2H), 2.36 (m, 4H), 1.92 (q, J ¼ 6:8 Hz, 2H). HRMS (EI) m/z:
calculated for C16H21N3O4, 319.1532; found, 319.1525.
A similar procedure was used to prepare 6-methoxy-7-[(1-methylpiperidin-
4-yl)methoxy]-3,4-dihydroquinazolin-4-one ð7cÞ. 1H NMR (360 MHz, DMSO-
d6): d : 7:98 (s, 1H), 7.43 (s, 1H), 7.11 (s, 1H), 3.97 (d, J ¼ 6:1 Hz, 2H), 3.87 (s,
3H), 2.78 (d, J ¼ 11:5 Hz, 2H), 2.15 (s, 3H), 1.86 (t, J ¼ 10:6 Hz, 2H), 1.74 (m,
3H), 1.32 (m, 2H). HRMS (EI) m/z: calculated for C16H21N3O3, 303.1583;
found, 303.1580.
Synthesis of 4-chloro-6,7-bis-(2-methoxyethoxy)-quinazoline ð8aÞ. 7a (0.5 g,
1.7 mmol) in CHCl3 (10 ml) containing one drop of DMF was added
oxalylchloride (720 mg, 5.7 mmol) in several portions over 5 min. Once
foaming ceased the solution was refluxed 1.5 h. The solvent was removed
under vacuum and the residue was dissolved in 1,2-dichloroethane (100 ml),
washed with saturated aqueous Na2CO3 (2 ꢁ 160 ml), dried over Na2SO4,
and concentrated under reduced pressure. Purification by flash chromato-
graphy (hexane/ethyl acetate from 10/0 to 4/6) afforded the solid title product
1
as a white powder (520 mg, 98%). H NMR (360 MHz, CDCl3): d : 8:86
(s, 1H), 7.44 (s, 1H), 7.33 (s, 1H), 4.34 (m, 4H), 3.89 (m, 4H), 3.50 (s, 3H), 3.49
(s, 3H). HRMS (EI) m/z: calculated for C14H17N2O4Cl, 312.0877; found,
312.0874.
A similar procedure was used to prepare 4-chloro-7-methoxy-6-(3-morpho-
linopropoxy)-quinazoline ð8bÞ and 4-chloro-6-methoxy-7-[(1-methylpiperidin-
1
4-yl)methoxy]-quinazoline ð8cÞ. 8b: H NMR (360 MHz, CDCl3): d : 8:87 (s,
1H), 7.40 (s, 1H), 7.33 (s, 1H), 4.28 (t, J ¼ 6:8 Hz, 2H), 4.05 (s, 3H), 3.74 (m,
4H), 2.60 (t, J ¼ 6:8 Hz, 2H), 2.51 (m, 4H), 2.14 (q, J ¼ 6:8 Hz, 2H). HRMS
(EI) m/z: calculated for C16H20N3O3Cl, 337.1193; found, 337.1192. 8c: H
1
NMR (360 MHz, DMSO-d6): d : 8:88 (s, 1H), 7.46 (s, 1H), 7.40 (s, 1H), 4.10
(d, J ¼ 6:1 Hz, 2H), 4.01 (s, 3H), 2.88 (d, J ¼ 11:2 Hz, 2H), 2.24 (s, 3H), 2.03
(m, 2H), 1.80 (m, 3H), 1.39 (m, 2H). HRMS (EI) m/z: calculated for
C16H20N3O2Cl, 321.1244; found, 321.1251.
Synthesis of 4-(30-chloro-40-fluoroanilino)-6,7-bis-(2-methoxyethoxy)-quinazo-
line ð9aÞ. A mixture of 8a (90 mg, 0.29 mmol), 3-chloro-4-fluoroaniline (46 mg,
0.32 mmol) in 2-propanol (1.5 ml) containing pyridine (25 ml) was heated at
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SYNTHESIS OF 18F-IRESSA AND RELATED MOLECULAR PROBES
839
reflux for 2 h. The volatiles were removed by evaporation. The residue was
taken in chloroform/methanol (9/1), washed with an aqueous solution of
NaHCO3 (5%), dried over Na2SO4, and concentrated under reduced pressure.
Purification by flash chromatography (CH2Cl2/MeOH from 10/0 to 19/1) gave
1
the solid title compound (65 mg, 53%). H NMR (360 MHz, DMSO-d6): d :
9:56 (s, 1H), 8.50 (s, 1H), 8.13 (dd, J ¼ 2:7 and 6.8 Hz, 1H), 7.85 (s, 1H), 7.79
(m, 1H), 7.46 (t, J ¼ 9:0 Hz, 1H), 7.24 (s, 1H), 4.28 (m, 4H), 3.75 (m, 4H), 3.37
(s, 3H), 3.35 (s, 3H). HRMS (MALDI-TOF) m/z: [M + H]+ calculated for
C20H22N3O4FCl+, 422.1277; found, 422.1257.
Synthesis of 4-(30-chloro-40-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)-
quinazoline ð9bÞ. A mixture of 8b (80 mg, 0.24 mmol), 3-chloro-4-fluoroaniline
(40 mg, 0.28 mmol) and DMF (2.5 ml) was stirred and heated to 1458C for 1 h.
The mixture was cooled to ambient temperature and the volatiles were
removed. The residue was applied to flash chromatography (CH2Cl2/MeOH
1
from 10/0 to 9/1) to give 9b (75 mg, 70%). H NMR (360 MHz, DMSO-d6):
d : 9:56 (s, 1H), 8.50 (s, 1H), 8.10 (m, 1H), 7.80 (m, 2H), 7.45 (t, J ¼ 9:2 Hz,
1H), 7.21 (s, 1H), 4.19 (t, J ¼ 6:1 Hz, 2H), 3.94 (s, 3H), 3.58 (m, 4H), 2.40 (m,
6H), 2.00 (m, 2H). HRMS (MALDI-TOF) m/z: [M + H]+ calculated for
C20H25N4O3FCl+, 447.1594; found, 447.1585.
Synthesis of 4-(30-chloro-40-fluoroanilino)-6-methoxy-7-[(1-methylpiperidin-
4-yl)methoxy]-quinazoline ð9cÞ. A mixture of 8c (60 mg, 0.19 mmol) and 3-
chloro-4-fluoroaniline (32.5 g, 0.22 mmol) in 2-propanol (3 ml) containing
concentrated HCl (17 ml) was heated at reflux for 2 h. The mixture was cooled
and the solid was filtered, washed with 2-propanol (2 ml) followed by ether
(5 ml) and dried under vacuum to yield 9c (71 mg, 88%). 1H NMR (360 MHz,
DMSO-d6): d : 9:80 (s, 1H), 8.49 (s, 1H), 8.19 (dd, J ¼ 2:9 and 6.9 Hz, 1H),
7.94 (s, 1H), 7.87 (m, 1H), 7.43 (t, J ¼ 9:2 Hz, 1H), 7.17 (s, 1H), 4.05–3.95 (m,
5H), 2.78 (d, J ¼ 10:4 Hz, 2H), 2.15 (s, 3H), 1.86 (t, J ¼ 11:2 Hz, 2H), 1.76 (m,
3H), 1.34 (m, 2H). HRMS (EI) m/z: calculated for C22H24N4O2ClF, 430.1572;
found, 430.1577.
Synthesis of 3-chloro-4-trimethylammonium-nitrobenzene trifluoromethanesul-
fonate ð10Þ. A solution of 2-chloro-4-nitroaniline (1.40 g, 8.1 mmol) in THF
(15 ml) was added under argon to NaH (60% suspension in mineral oil, 0.7 g,
17.5 mmol). The reaction mixture was stirred 5 min at room temperature under
argon, followed by the addition of a solution of iodomethane (2.84 g, 20 mmol)
in THF (5 ml). After the solution was stirred for 24 h at ambient temperature,
the reaction mixture was poured into cold water. The mixture was diluted with
ethyl acetate and the organic layer was separated. The aqueous layer was
further extracted with ethyl acetate, and the organic layers were combined.
The organic layers were washed with water, brine, dried over Na2SO4, and
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concentrated under reduced pressure. The residue was applied to flash
chromatography (Hexanes/CHCl3 from 10/0 to 7/3) to give 3-chloro-4-
1
(dimethylamino)nitrobenzene (1.35 g, 83%). H NMR (360 MHz, CDCl3): d :
8:22 (s, 1H), 8.06 (d, J ¼ 8:3 Hz, 1H), 6.97 (d, J ¼ 8:3 Hz, 1H), 3.01 (s, 6H). 3-
Chloro-4-(dimethylamino)nitrobenzene (0.6 g, 3.0 mmol) was dissolved
CH2Cl2 (5 ml) and treated with methyl trifluoromethanesulfonate (0.99 g,
6.0 mmol) for 24 h at room temperature under an argon atmosphere. The
mixture was diluted with diethyl ether and the solid residue was filtered off.
The residue was recrystallized twice from CH2Cl2/diethyl ether to give the title
1
compound (0.89 g, 80%) as white needles. H NMR (360 MHz, Acetone-d6):
d : 8:62 (m, 2H), 8.47 (dd, J ¼ 2:7 and 9.6 Hz, 1H), 4.20 (s, 9H).
Radiochemistry
Preparation of 3-chloro-4-[18F]fluoroaniline ð12Þ. A 5 ml V-vial was loaded
with 0.5 ml of a CH3CN/H2O solution of potassium carbonate (0.5 mg,
3 mmol) and Kryptofix [2.2.2] (K222, 2.25 mg, 6 mmol). Fluorine-18 in water was
added and the V-vial was gently heated at 1108C. Water was removed under a
stream of argon by azeotropic distillation with acetonitrile (3 ꢁ 0.5 ml) to give
the no-carrier-added K[18F]F-K222 complex as a white semi-solid residue. A
solution of 3-chloro-4-trimethylammonium-nitrobenzene triflate (10, 2.2 mg,
6 mmol) in anhydrous acetonitrile or dimethyl sulfoxide (0.5 ml) was added into
the vial containing dry cryptate (K[18F]F-K222) and stirred under the given
conditions described in Table 1. After cooling the mixture was poured into
water (8 ml), and the aqueous solution was passed through a C18 Sep-Pak.
The Sep-Pak was rinsed with additional water (5 ml) and 3-chloro-4-
[18F]fluoro-nitrobenzene ð11Þ was eluted through the column with MeOH
(1.5 ml) into a tube containing sodium borohydride (7 mg, 0.18 mmol) and
palladium on activated carbon (10%, 1 mg). The methanolic solution was
stirred at room temperature for 5 min, and the reaction was quenched with
concentrated HCl (0.2 ml). The solution was subsequently filtered, and the
filter was washed with MeOH (0.5 ml). Evaporation of the volatiles under
reduced pressure gave 3-chloro-4-[18F]fluoroaniline ð12Þ. Typically, 1.4–
2.2 GBq (38–60 mCi) of 3-chloro-4-[18F]fluoroaniline could be obtained in
70–80 min starting from 3.4 to 6.1 GBq (92–165 mCi) aliquot of a cyclotron-
produced [18F]Fꢀ batch (overall radiochemical yields, based on starting
[18F]fluoride: 58–72% decay corrected).
Radiosynthesis of 4-(30-chloro-40-[18F]fluoroanilino)-6,7-bis-(2-methoxyethoxy)-
quinazoline ([18F]-9a). A solution of 8a (1 mg, 3 mmol) in 2-propanol (2 ml)
was added to the dry 3-chloro-4-[18F]fluoroaniline ð12Þ and stirred at 858C for
15min. The volatiles were removed under pressure, and the residue was
reconstituted into a (1/1) mixture of MeOH/phosphate buffer at pH 5.8. [18F]-9a
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SYNTHESIS OF 18F-IRESSA AND RELATED MOLECULAR PROBES
841
was purified by HPLC (Phenomenex, Luna-C18, 5mm, 250 mm ꢁ 10mm) with
a linear gradient (0–15min) of 50–100% MeOH in phosphate buffer (2ml/min)
followed by 15min isochratic elution with 100% MeOH. Retention time of
[18F]-9a was 22 min vs 15 and 17 min for the starting materials 12 and 8a,
respectively, under the same elution conditions. Typically, 0.4–0.6 GBq (12–
16 mCi) of pure [18F]-9a could be obtained in 50–55 min starting from 1.4–
1.5 GBq (38–41 mCi) of 3-chloro-4-[18F]fluoroaniline (overall radiochemical
yields, based on starting 3-chloro-4-[18F]fluoroaniline: 43–55% decay corrected).
Radiosynthesis of 4-(30-chloro-40-[18F]fluoroanilino)-7-methoxy-6-(3-morpho-
linopropoxy)quinazoline ([18F]-9b). A solution of 8b (1 mg, 3 mmol) in 2-
propanol (2 ml) was added to the dry 3-chloro-4-[18F]fluoroaniline ð12Þ and
stirred at 1208C for 15 min. The volatiles were removed under pressure, and
the residue was reconstituted into a (1/1) mixture of MeOH/phosphate buffer
at pH 5.8. [18F]-9b was purified by HPLC (Phenomenex, Luna-C18, 5 mm,
250 mm ꢁ 10 mm) with a linear gradient (0–15 min) of 50–100% MeOH in
phosphate buffer (2 ml/min) followed by 15 min isochratic elution with 100%
MeOH. Retention time of [18F]-9b was 21 min vs 17 min for the starting
material 8b under the same elution conditions. Typically, 0.7–1.1 GBq (19–
30 mCi) of pure [18F]-9b could be obtained in 50–55 min starting from 1.4–
2.2 GBq (38–60 mCi) of 3-chloro-4-[18F]fluoroaniline (overall radiochemical
yields, based on starting 3-chloro-4-[18F]fluoroaniline: 60–70% decay
corrected).
Radiosynthesis of 4-(30-chloro-40-[18F]fluoroanilino)-6-methoxy-7-[(1-methyl-
piperidin-4-yl)methoxy]-quinazoline ([18F]-9c). A solution of 8c (1 mg,
3 mmol) in 2-propanol (2 ml) was added to the dry 3-chloro-4-[18F]fluoroaniline
ð12Þ and stirred at 1208C for 15 min. The volatiles were removed under
pressure, and the residue was reconstituted into a (1/1) mixture of MeOH/
phosphate buffer at pH 4.7. [18F]-9c was purified by HPLC (Phenomenex,
Luna-C18, 5 mm, 250 mm ꢁ 10 mm) with a linear gradient (0–15 min) of 50–
100% MeOH in phosphate buffer (2 ml/min) followed by 15 min isochratic
elution with 100% MeOH. Retention time of [18F]-9c was 18 min vs 15 and
11 min for the starting materials 12 and 8c, respectively, under the same
elution conditions. Typically, 0.6 GBq (15–17 mCi) of pure [18F]-9c could be
obtained in 50–55 min starting from 1.4 to 1.7 GBq (38–45 mCi) of 3-chloro-4-
[18F]fluoroaniline (overall radiochemical yields, based on starting 3-chloro-4-
[18F]fluoroaniline: 46–56% decay corrected).
Formulation of the 18F labeled TKI ([18F]-9a2c) and quality control. HPLC
fractions (4–5 ml) containing the 18F labeled product were collected and
solvents were evaporated under reduced pressure. The tracer was dissolved in
ethanol, and the solution was then diluted to the proper dosage with sterile
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Y. SEIMBILLE ET AL.
physiological saline, so that the injection dose contained no more than 10% of
alcohol.
As demonstrated by HPLC analysis (Phenomenex Luna C-18 column,
10 ꢁ 250 mm, 5 mm), radiolabeled anilinoquinazoline derivatives ([18F]-9a–c)
co-eluted with authentic synthesized unlabeled reference 9a–c. The radio-
pharmaceuticals were found to be >95% chemically and radiochemically
pure.
Conclusion
In conclusion, this report describes a synthetic strategy for the production of a
wide variety of radiolabeled 6,7-disubstituted 4-anilinoquinazolines suitable
for imaging tyrosine kinase receptors to predict therapy effectiveness. Three
new F-18 labeled radiopharmaceuticals based on the therapeutic agents
Tarceva, Iressa, and ZD6474 were synthesized. Conditions for radiosynthesis
are suitable for preparation of the compounds in quantities and times which
are practical for use as PET radiopharmaceuticals. Cell uptake studies and
preliminary microPET imaging in disease-free and tumor bearing animals
have been performed, and further evaluation are underway to demonstrate the
feasibility of using those F-18 labeled tyrosine kinase inhibitors as potential
imaging agents to predict efficacy of those antitumor therapeutics in vivo.
Acknowledgements
This work was supported in part by California Breast Cancer Research
Program Grant 7KB-0155, and DOE DE-FC03-87ER60615. We thank the
cyclotron staff at UCLA and the UCLA Pasarow Mass Spectrometry
Laboratory for their collaboration.
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