Metabolic stability of 6,7-dialkoxy-4-(2-, 3- and 4-[18F] fluoroanilino)quinazolines, potential EGFR imaging probes

Article abstract of DOI:10.1016/j.bmc.2011.03.032

Epidermal growth factor receptors (EGFR), upregulated in many tumor types, have been a target for therapeutic development and molecular imaging. The objective of this study was to evaluate the distribution and metabolic characteristics of fluorine-18 labe

Full text of DOI:10.1016/j.bmc.2011.03.032

Bioorganic & Medicinal Chemistry 19 (2011) 2959–2965
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Metabolic stability of 6,7-dialkoxy-4-(2-, 3- and
4-[18F]fluoroanilino)quinazolines, potential EGFR imaging probes
Neil Vasdev a, Peter N. Dorff a, James P. O’Neil a, Frederick T. Chin a, Stephen Hanrahan a,
Henry F. VanBrocklin a,b,
⇑
a Department of Radiotracer Development and Imaging Technology, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States
b Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, CA 94107, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Epidermal growth factor receptors (EGFR), upregulated in many tumor types, have been a target for ther-
apeutic development and molecular imaging. The objective of this study was to evaluate the distribution
and metabolic characteristics of fluorine-18 labeled anilinoquinazolines as potential imaging agents for
EGFR tyrosine kinase expression. Fluorine-18 labeled fluoronitrobenzenes were prepared by reaction of
potassium cryptand [18F]fluoride with 1,2- and 1,4-dinitrobenzenes, and 3-nitro-N,N,N-trimethylanilini-
um triflate in 5 min. Decay-corrected radiochemical yields of [18F]fluoride incorporation into the nitro-
aromatic compounds were 81 2%, 44 4% and 77 5% (n = 3–5) for the 2-, 3- and 4-fluoro isomers,
respectively. Sodium borohydride reduction to the corresponding [18F]fluoroanilines was achieved with
greater than 80% conversion in 5 min. Coupling of [18F]fluoroaniline-hydrochlorides to 6,7-dimethoxy-
4-chloro-quinazoline gave the corresponding 6,7-dimethoxy-4-(2-, 3- and 4-[18F]fluoroanilino)
quinazolines in 31 5%, 17 2% and 55 2% radiochemical yield, respectively, while coupling to the
6,7-diethoxy-4-chloro-quinazoline produced 6,7-diethoxy-4-(2-, 3- and 4-[18F]fluoroanilino)quinazo-
lines in 19 6%, 9 3% and 36 6% radiochemical yield, respectively, in 90 min to end of synthesis from
Received 13 January 2011
Revised 7 March 2011
Accepted 14 March 2011
Available online 21 March 2011
Keywords:
PET
Fluorine-18
Fluoroaniline
Metabolism
Quinazoline
EGFR
Hepatocytes
[
18F]fluoride. Biodistribution of 2- and 4-[18F]fluoroanilinoquinazolines was conducted in tumor-bearing
mice (MDA-MB-435 and MDA-MB-468 xenografts). Low tumor uptake (<1% injected dose per gram (ID/g)
of tissue up to 3 h postinjection of the radiotracers) was observed. High bone uptake (5–15% ID/g) was
noted with the 4-[18F]fluoroanilinoquinazolines. The metabolic stabilities of radiolabeled quinazolines
were further evaluated by incubation with human female cryopreserved isolated hepatocytes. Rapid
degeneration of the 4-fluoro-substituted compounds to baseline polar metabolites was observed by
radio-TLC, whereas, the 2- and 3-[18F]fluoroaniline derivatives were significantly more stable, up to
2 h, corroborating the in vivo biodistribution studies. para-Substituted [18F]fluoroanilines, a common
structural motif in radiopharmaceuticals, are highly susceptible to metabolic degradation.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
in a variety of tumors is often an indicator of poor prognosis.2
EGFR, as well as other ErbB family members, are established bio-
The design of potent and selective protein kinase inhibitors is a
major goal of drug discovery programs and within the last decade
several small molecule kinase inhibitors have been approved for
clinical use.1 The epidermal growth factor receptor (EGFR) is a
member of the ErbB family of receptor tyrosine kinases, cell surface
receptors that activate a variety of cellular events including cell
proliferation and differentiation. Overexpression of these receptors
markers and targets for cancer therapy.3–5 Of the therapeutic ap-
proaches taken to develop EGFR-based drugs, one of the most
actively pursued involves small molecule tyrosine kinase inhibitors
that compete with the adenosine triphosphate (ATP)-binding pock-
et of the cytoplasmic domain of the receptor, inhibiting receptor
phosphorylation and blocking the transduction of downstream sig-
naling cascades.6 The first drug candidates, 6,7-dimethoxy-4-(3-
bromoanilino)quinazoline7,8 (PD153035; Fig. 1) and its diethoxy
derivative, were very potent and selective inhibitors of EGFR tyro-
sine kinase activity (IC50 = 25 and 6 pM, respectively). Subse-
quently, several anilinoquinazoline-based EGFR inhibitors have
been evaluated, with Gefitnib (ZD1839, Iressa, AstraZeneca) and
Erlotinib (Tarceva, OSI-774, Genentech and OSI Pharmaceuticals;
Fig. 1) achieving clinical use approval by the US FDA.9
⇑
Corresponding author. Address: Professor of Radiology and Biomedical Imaging,
University of California San Francisco, Director of Radiopharmaceutical Research,
Center for Molecular and Functional Imaging, 185 Berry Street, Suite 350, San
Francisco, CA 94107, United States. Tel.: +1 415 353 4569; fax: +1 415 514 8242.
E-mail addresses: henry.vanbrocklin@ucsf.edu, henry.vanbrocklin@radiolo-
gy.ucsf.edu (H.F. VanBrocklin).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.032

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N. Vasdev et al. / Bioorg. Med. Chem. 19 (2011) 2959–2965
F
HN
N
Br
O
HN
N
Cl
HN
N
H3CO
H3CO
N
O
O
O
N
N
H3CO
H3CO
N
H3CO
PD153035
Gefitinib (Iressa; ZD1839)
Erlotinib (Tarceva; OSI-774)
Figure 1. Quinazoline-based inhibitors of the EGFR tyrosine kinase.
18F
Cl
N
N
18F
18F
R1O
R1O
R
HN
N
K18F/K222
1. NaBH , Pd-C
2. HCl
HPLC
R1O
R1O
N
DMSO, Δ
DMF, Δ
HCl
NO2
NO2
NH2
R = NO2 (ortho, para); R = Me3N+ OTf - (meta)
1: R1 = CH3
a: 18F = 2'
b: 18F = 3'
c: 18F = 4'
2: R1 = CH3CH2
Scheme 1. Syntheses of [18F]1 and [18F]2 via [18F]fluoroanilines.
An effective single photon emission computed tomography
(SPECT) or positron emission tomography (PET) imaging agent
for the EGFR could facilitate the development of therapeutics and
provide improved criteria for patient selection.10,11 Consequently,
targeting of the extracellular EGFR domain with radiolabeled
monoclonal antibodies, affibodies or peptides, or employing radio-
labeled small molecules to target the intracellular kinase domain
has emerged as a major molecular imaging initiative. This field
has been extensively reviewed by several laboratories,12–21 includ-
ing ours.22 The majority of radiolabeled EGFR tyrosine kinase
inhibitors are 4-anilino-quinazoline derivatives, based on the pro-
totypical reversibly binding inhibitor, PD153035. Recently, [11C]-
PD153035 has advanced to preliminary human studies.23 The short
half-life of carbon-11 (20.4 min) may limit the widespread use of
this radiopharmaceutical to sites that have an on-site cyclotron,
and may not allow adequate time for the radiotracer to accumulate
in tumor cells that overexpress EGFR. In an attempt to overcome
the limitations of the short half-life of carbon-11, we24 and several
other laboratories22 have directed our research efforts towards
preparing fluorine-18 (half-life = 109.7 min) labeled EGFR tyrosine
kinase inhibitors that may achieve adequate tumor to background
contrast.
The metabolism of pharmaceutical compounds is important for
the delivery and availability of the molecules to reach the intended
target site in vivo. Likewise radiopharmaceutical metabolism is
also a critical parameter for the development of new tracers. Spe-
cies-specific metabolism precludes the prediction of compound
metabolism in humans. Isolated hepatocytes have been applied
to evaluate the metabolism of radiopharmaceuticals in several spe-
cies and can be used to evaluate a series of compounds in the same
species.25 Hepatocyte assays may be used to portend the metabolic
fate of short-lived radiotracers in humans, providing an important
parameter for tracer development.
radiolabeled for PET imaging in an array of in vitro assays.26 This
report identified two compounds that warrant radioisotope label-
ing with fluorine-18, namely, 4-(2-fluoroanilino)- and 4-(3-fluoro-
anilino)-6,7-diethoxyquinazoline, for further evaluation in vivo.
The goals of the present work are: (1) to efficiently synthesize
6,7-dimethoxy- and 6,7-diethoxy-4-(2-, 3- and 4-[18F]fluoroanili-
no)quinazolines; (2) to evaluate the imaging potential of select
compounds in tumor-bearing mice; and (3) to investigate the
metabolic stability of these radiotracers in human hepatocytes
compared to the in vivo mice distribution data.
2. Results and discussion
2.1. Radiochemistry
In order to investigate the positional effect of the fluorine-18
bound to the [18F]fluoroanilino-group, our first goal was to opti-
mize the preparation of 2-, 3- and 4-[18F]fluoroanilines for rapid
coupling to the respective 4-chloro-quinazoline derivatives, to pre-
pare the title radiotracers. The preparation of fluorine-18 labeled
dialkoxy-fluoroanilinoquinazolines is shown in Scheme 1. Interme-
diates, 2- and 4-[18F]fluoronitrobenzene, were produced by reac-
tion of potassium
[
18F]fluoride/cryptand complex (K18F/
KryptofixÒ (K222)) with the corresponding 1,2- and 1,4-dinitro-
benzenes, and 3-[18F]fluoronitrobenzene was prepared from 3-ni-
tro-N,N,N-trimethylanilinium triflate. The radiochemical yields, as
well as the respective time course for these syntheses, are shown
in Table 1.
The syntheses of 2- and 4-[18F]fluoronitrobenzene was effi-
cient, consistent with previous work.27–30 The incorporation of
[
18F]fluoride into the 1,3-substituted dinitrobenzene has histori-
cally been a low yielding (<17%) reaction.27 The use of 3-nitro-
N,N,N-trimethylanilinium triflate gave 3-[18F]fluoronitrobenzene
We previously reported a systematic study that screened
several EGFR tyrosine kinase inhibitors that could be readily
in reasonable yield (44 4%) after 5 min using
a modified

N. Vasdev et al. / Bioorg. Med. Chem. 19 (2011) 2959–2965
2961
method.31 A competing side reaction of [18F]fluoride ion with one
of the methyl groups on the trimethylammonium moiety pro-
duces [18F]fluoromethane.32
Table 1
Time course and radiochemical yields during [18F]fluoroanilinoquinazoline syntheses
Compound
Time coursea
(min)
% Radiochemical yieldb
The [18F]fluoronitrobenzenes were purified by C18 solid phase
extraction (SPE) and reduced to the corresponding [18F]fluoroani-
lines in 5 min at room temperature using a modification of a pre-
viously reported method (Scheme 1).33 The reduction reaction
was quenched with excess HCl to give the [18F]fluoroaniline-
hydrochloride salt. Formation of the salt was necessary to prevent
the complete loss of the [18F]fluoroaniline during the solvent evap-
oration process.
a (ortho-F) b (meta-F) c (para-F)
[
[
[
[
18F]Fluoronitrobenzenec 20
81
68
31
19
2
2
5
6
44
35
17
9
4
3
2
3
77
72
55
36
5
6
2
6
18F]Fluoroaniline
18F]1 (dimethoxy-)
18F]2 (diethoxy-)
30
90d
90d
a
100% of activity at time of 0 min is attributed to [18F]Fluoride and represents the
start of synthesis.
b
Decay-corrected relative to [18F]fluoride; n = 3–5.
Precursor: 1,2- and 1,4-dinitrobenzene (a and c); 3-nitro-N,N,N-trimethylani-
c
Coupling the [18F]fluoroanilines to 6,7-dimethoxy- or 6,7-dieth-
oxy-4-chloro-quinazoline yielded [18F]1a–c and [18F]2a–c, respec-
tively, as shown in Scheme 1. Moderate yields were obtained for
linium triflate (b).
d
Following preparative HPLC and sample concentration/formulation.
100
a
90
80
70
60
50
40
30
20
10
0
[
18F]1b
[
18F]1a
[
18F]1c
0
20
40
60
80
100
120
Time (min)
b
100
90
80
70
60
50
40
30
20
10
0
[
18F]2b
[
[
18F]2a
18F]2c
0
20
40
60
80
100
120
Time (min)
Figure 2. Metabolic stability of (a) [18F]1 and (b) [18F]2 by female human hepatocytes over 2 h.

2962
N. Vasdev et al. / Bioorg. Med. Chem. 19 (2011) 2959–2965
the coupled quinazolines ([18F]1a–c and [18F]2a–c; Table 1). The
yields for the dimethoxy-derivatives ([18F]1a–c) were consistently
greater than the respective diethoxy-compounds ([18F]2a–c). The
relatively low measured specific activities of 44–308 Ci/mmol
(1.5–11.4 MBq/mmol) are attributed to the small initial amounts
of fluoride used (2.5–6.5 mCi; 93–241 MBq) for the synthesis.
Greater than 100 mCi (3.7 GBq) of starting [18F]-fluoride was used
for all compounds prepared for biological evaluations and specific
time consuming syntheses reported herein enabled biological eval-
uations of [18F]1b to be carried out for the first time.
2.2. Biological studies
2.2.1. Metabolism of [18F]-fluoroanilinoquinazolines by human
hepatocytes
Species differences in metabolism are well-known. This phe-
nomenon has been seen with radiopharmaceuticals as well.35 The
distribution differences among the positional isomers seen in the
current mouse study (see Section 2.2.2) and the previously
reported degradation of the 4-[18F]fluoroanilinoquinazline34
prompted a further evaluation of the metabolic fate of these
activities correspond to >1 Ci/lmol at the time of injection. The to-
tal synthesis time for the combined reactions was optimized at
90 min. Our yields improved upon the previously reported34 non-
decay corrected radiochemical yield for compounds [18F]1b and
[
18F]1c (ca. 1% and 4–12%, respectively). The more facile and less
2.0
a
1.5
1.0
0.5
0.0
1 hr
3 hr
Blood
Heart
Lung
Liver
Kidney
Spleen
Bone
Muscle
MB-468 MB-435
20
4
b
3
1 hr
2 hr
2
1
0
Blood Heart Lung Liver Kidney Spleen Brain Bone Muscle Fat MB-468
Figure 3. Ex vivo biodistribution of (a) [18F]1a, (b) [18F]1c, (c) [18F]2a, and (d) [18F]2c in tumor-bearing mice.

N. Vasdev et al. / Bioorg. Med. Chem. 19 (2011) 2959–2965
2963
5
4
3
2
1
0
c
1 hr
3 hr
Blood
Heart
Lung
Liver
Kidney Spleen
Bone
Muscle MB-468 MB-435
8
d
7
6
5
4
3
2
1
0
1 hr
2 hr
Blood
Heart
Lung
Liver
Kidney Spleen
Brain
Bone
Muscle
Fat
MB-468
Fig. 3 (continued)
compounds. Human hepatocytes were chosen in order to deter-
mine whether the same metabolic pathways active in the mouse
were present in humans.
The rapid metabolism of 4-[18F]fluoroanilinoquinazolines in the
present and previous work34,36 is likely due to defluorination.
These results are consistent with reported defluorination of 19F
4-fluoroaniline-containing compounds.37–39 The 2-[18F] and 3-[18F]
fluoroanilinoquinazolines are more stable in the hepatocyte assay
suggesting that they are more suitable for in vivo imaging.
The six [18F]-fluoroanilinoquinazolines were added to human
hepatocyte preparations and the time course of tracer metabolism
over 120 min was monitored by radio-TLC on aliquots of the med-
ia. The metabolic degradation of [18F]1a–c and [18F]2a–c is shown
in Figure 2a and b, respectively. The pattern of parent radiotracer
loss over time, 4-[18F]fluoro > 2-[18F]fluoro > 3-[18F]fluoro, was
similar for both series. The 4-[18F]fluoroanalino compounds in both
series showed comparable rapid exponential metabolism with
complete consumption of the tracer within ꢀ90 min. The dime-
thoxy-2-[18F]fluoro and 3-[18F]fluoro analogs were consumed
more slowly than the corresponding diethoxy derivatives. All of
the degradation products were more polar than the parent radio-
tracer with the majority of activity at the baseline. Thorough char-
acterization of the polar metabolites was not conducted.
2.2.2. Ex vivo biodistribution studies in tumor-bearing mice
The 2-[18F]- and 4-[18F]-fluoroanilinoquinazolines were se-
lected for evaluation in the mouse xenograft models as they were
prepared in higher radiochemical yields compared to the 3-[18F]
analogs. Ex vivo biodistribution studies were carried out in mice
with both EGFR positive (MDA-MB-468) and EGFR negative
(MDA-MB-435) or only EGFR positive (MDA-MB-468) xenografts.
The tumors, normal tissues, blood and bone were excised and
counted for radioactivity from 1 to 3 h postinjection of the radio-
tracers. The organ distributions for all four compounds is shown

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N. Vasdev et al. / Bioorg. Med. Chem. 19 (2011) 2959–2965
in Figure 3. These compounds showed limited tumor uptake (<0.6%
injected dose (ID) per gram of tumor) at all time points. There was
no difference in uptake between the EGFR positive and EGFR neg-
ative xenografts. A blocking study was carried out with [18F]1a by
pre-injection of PD153035 (1 mg/kg administered 1 h prior to radi-
otracer injection). The pre-blocking failed to reduce radiotracer up-
take compared to the control group.
nium trifluoromethanesulfonate was synthesized as previously
described.31
4.1.2. Standard techniques
No-carrier-added 18F-fluoride was produced by 10 MeV proton
irradiation of 30% enriched [18O]H2O in a silver target by the
18O(p,n)18F nuclear reaction on the LBNL Biomedical Isotope Facil-
Bone uptake for the 2-[18F] analogs (Fig. 3a,c) was less than 2%
ID/g at all time points. The 4-[18F] analogs (Fig. 3b,d) demonstrated
significantly greater bone uptake, 5–15% ID/g. The high bone accu-
mulation of these compounds is indicative of in vivo defluorination
and corroborates the hepatocyte studies that also show a rapid
degradation of these compounds to polar species, likely [18F]fluo-
ride. Rapid metabolism of [18F]1c was previously noted yet the
bone uptake was not analyzed in that study.34 This further demon-
strates the general metabolic instability of 4-fluoroanilino com-
ity CTI RDS 111 cyclotron. The [18O]H2O/18FÀ (ca. 100–450
l
L) was
conical glass vial containing K222 (5 mg,
mol), K2CO3 (0.5 mg, 3.6 mol) in 500 L of CH3CN. The water
transferred into
13
a
l
l
l
was removed by azeotropic distillation with anhydrous CH3CN
(1 mL) at 100 °C in vacuo under a stream of nitrogen. The drying
process was repeated.
4.1.3. Synthesis of [18F]fluoroanilines
The conversion of [18F]fluoronitrobenzenes to [18F]fluoroani-
lines described here is a modification of previously reported
pounds and serves as
a cautionary note for its use in
pharmaceutical and radiopharmaceutical design. It is also interest-
ing to note that this metabolic defluorination process is active in
assays from different species.
methods.24,33 The 1,2- or 1,4-dinitrobenzene (ca. 0.5 mg, 3
l
mol),
or 1-nitro-3-aryltrimethylammonium trifluoromethanesulfonate
(ca. 0.5 mg, 2 mol) was dissolved in 250 L of DMSO and trans-
l
l
‘‘The fluorine effect’’ describes the phenomenon that occurs
when differences in pharmacological properties between fluori-
nated and non-fluorinated derivatives of a compound are dis-
played, and is attributed to the specific site of fluorine
substitution.40,41 The specific site of aryl ring fluorination with
18F is well-known to alter pharmacokinetic properties of radio-
pharmaceuticals in vivo. For example, an evaluation of aromatic
ferred into a conical vial containing the dried K18F/K222. The vessel
was sealed with a Teflon-faced silicone septum and heated at
130 °C for 5 min. The sample was diluted to 5 mL with water and
loaded onto an activated (MeOH) and equilibrated (water) C18
Sep-PakÒ cartridge (Waters). The cartridge was flushed with 4 mL
of water and the [18F]fluoronitrobenzene was eluted with 2 mL of
MeOH directly into a flat-bottomed glass vial (4 mL) containing
ring-substituted 2-, 5- or 6-[18F]fluoro-
([18F]fluoro-
L
-dihydroxyphenylalanine
NaBH4 (10 mg, 260
was allowed to proceed for 5 min at room temperature prior to
quenching with 100 L of concentrated HCl. The solution was
lmol) and 10 wt % Pd-C (4 mg). The reduction
L-DOPA) revealed dramatically different biological
properties in human imaging studies.42 Therefore, it is not surpris-
ing that 2-, 3- and 4-substituted [18F]fluoroanilinoquinazolines
also demonstrated different biological properties in vivo. This
study further illustrates the importance of isomeric considerations
in radiopharmaceutical design.
l
filtered through Celite, then evaporated to dryness at 100 °C in
vacuo with the aid of CH3CN.
4.1.4. Synthesis of [18F]fluoroanilinoquinazolines
The dried [18F]fluoroaniline-hydrochlorides were coupled to
6,7-diethoxy- (2.2 mg, 9
l
mol) or 6,7-dimethoxy-4-chloro-quinaz-
3. Conclusions
oline (2.2 mg, 10 mol) in 250
l
lL of DMF and reacted at 100 °C for
15 min. The reaction mixture was made basic with 1 N NaOH
(1 mL) and loaded onto an activated C18 Sep-PakÒ cartridge. The
cartridge was flushed with 4 mL of H2O, eluted with 2 mL of MeOH
and filtered through Celite and a 0.45 mm nylon membrane filter
prior to preparative HPLC purification.
Fluorine-18 labeled 2-, 3- and 4-fluoroanilines were prepared as
key intermediates in the synthesis of a series of 6,7-dimethoxy-
and 6,7-diethoxy-4-(2-, 3- and 4-[18F]fluoroanilino)quinazolines
in yields suitable for further study as EGFR imaging agents. Rapid
degradation of 6,7-dialkoxy-4-(4-[18F]fluoroanilino)quinazolines
was seen in the in vitro human hepatocyte assay as well as by high
bone uptake in mouse studies. This is consistent with metabolic
defluorination in this class of compounds. As 4-[18F]fluoroanilines
are extensively used in PET radiopharmaceutical development, this
work shows the importance of the position of the radiolabel on the
in vivo behavior of [18F]-fluoroaniline-based radiopharmaceuticals
in multiple species.
The reaction mixtures were purified by preparative HPLC puri-
fication (column; Beckman Ultrasphere ODS-5
l
m, 10 mm Â
25 cm, mobile phase; 65:35 (v/v) CH3OH: (1% Et3N in H2O adjusted
to pH 7.4 with H3PO4), flow rate; 4 mL minÀ1 ([18F]1a–c),
6 mL minÀ1 ([18F]2a–c)). The column eluent was monitored for
UV absorbance at 254 nm (UV detector: Linear, UV 106) and radio-
activity (detector: Carroll and Ramsey Associates, Model 105S). All
[
18F]fluoroanilinoquinazolines were identified by HPLC via co-
injection of an aliquot of the final product with authentic sample,
prepared as previously described by our group.26 Compounds
4. Experimental section
4.1. Radiochemistry
[
18F]1a–c were isolated at a flow rate of 4.0 mL minÀ1 and had
retention times of 7, 13 and 10.5 min, respectively. Compounds
[
18F]2a–c were isolated at a flow rate of 6 mL minÀ1 and had reten-
tion times of 9.5, 12 and 14.5 min, respectively. Compounds [18F]1
and [18F]2 had high radiochemical purities (>95%). Specific activi-
ties were determined by comparing the integration of the HPLC
peak area to a standard curve generated by an authentic sample
of the compound at various concentrations.
4.1.1. Materials
Anhydrous acetonitrile, dimethyl sulfoxide and N,N-dimethyl-
formamide were purchased from Aldrich (99.8%) and used without
further purification. KryptofixÒ 222 (Aldrich, 98%), K2CO3 (Aldrich,
>99%) NaBH4 (Spectrum), palladium, 10 wt % on activated carbon
(Aldrich), HCl (Aldrich), absolute EtOH USP (Aaper), HPLC grade
MeOH (Burdick & Jackson) and [18O]H2O (Rotem Industries, Ltd,
Israel, >94.1 atom%) were used without further purification. The
1,4-dinitrobenzene (Aldrich, 98%) and 1,2-dinitrobenzene (>99%)
compounds were purchased from Aldrich and recrystallized from
ethyl acetate prior to use, while and 1-nitro-3-aryltrimethylammo-
4.2. Biological studies
4.2.1. Metabolic studies with hepatocytes
2-, 3- or 4-[18F]Fluoroanilino-6,7-dialkoxyquinazoline were inde-
pendently synthesized and dissolved in modified Krebs–Henseleit

N. Vasdev et al. / Bioorg. Med. Chem. 19 (2011) 2959–2965
2965
buffer. Cryopreserved female human hepatocytes (In Vitro Tech-
References and notes
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pended in the buffer and divided into 4 wells of a 12 well
plate. The buffer solution containing the 2-, 3- or 4-[18F]fluoroani-
lino-6,7-dialkoxyquinazolines (0.5 mL) was transferred into three
of the wells. The prepared cells were maintained at 37 °C in a CO2
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Acknowledgements
We thank Dr. Andrew Gibbs and Dr. Erathodiyil Nandanan for
their synthetic expertise and helpful discussions. This work was
supported by the Director, Office of Science, Office of Biological
and Environmental Research, Medical Sciences Division of the
U.S. Department of Energy under Contract No. DE-AC03-
76SF00098 and NIH grant #CA94253 (HFV) and #CA79823 (HFV).
This work was also sponsored by the Department of the Army un-
der Grant No. DAMD17-98-1-8064 (HFV) and the State of Califor-
nia under Grant No. UCBCRP 4IB-0059 (HFV). We thank the
Natural Sciences and Engineering Research Council of Canada for
a Postdoctoral Fellowship (N.V.). We also thank Chris Ramsey for
technical expertise and production of [18F]fluoride.
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