Radiosynthesis and biological evaluation of L- and D-S-(3-[ 18F]fluoropropyl)homocysteine for tumor imaging using positron emission tomography

Article abstract of DOI:10.1021/jm101513q

Interest in radiolabeled amino acids for metabolic imaging of cancer and limitations with [¹¹C]methionine has prompted the development of a new ¹⁸F-labeled methionine derivative S-(3-[¹⁸F] fluoropropyl)homocysteine ([

Full text of DOI:10.1021/jm101513q

ARTICLE
pubs.acs.org/jmc
Radiosynthesis and Biological Evaluation of L- and
D-S-(3-[18F]Fluoropropyl)homocysteine for Tumor
Imaging Using Positron Emission Tomography
Thomas Bourdier,*,† Rachael Shepherd,† Paula Berghofer,† Timothy Jackson,† Christopher J. R. Fookes,†
Delphine Denoyer,‡ Donna S. Dorow,‡ Ivan Greguric,† Marie-Claude Gregoire,† Rodney J. Hicks,‡ and
Andrew Katsifis†
†ANSTO LifeSciences, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC, NSW, 2232,
Sydney, Australia
‡Centre for Molecular Imaging, The Peter MacCallum Cancer Centre, 12 St. Andrew's Place, East Melbourne, VIC, 3002, Australia
S Supporting Information
b
ABSTRACT: Interest in radiolabeled amino acids for metabolic
imaging of cancer and limitations with [11C]methionine has
prompted the development of a new 18F-labeled methionine
derivative S-(3-[18F]fluoropropyl)homocysteine ([18F]FPHCys).
The L and D enantiomers of [18F]FPHCys were prepared from
their respective protected S-(3-tosyloxypropyl)homocysteine
precursors 1 by [18F]fluoride substitution using K2.2.2 and
potassium oxalate, followed by acid hydrolysis on a Tracerlab
FXFN synthesis module. [18F]-L-FPHCys and [18F]-D-FPHCys
were isolated in 20 ( 5% radiochemical yield and >98%
radiochemical and enantiomeric purity in 65 min. Competitive uptake studies in A375 and HT29 tumor cells suggest that
L- and D-[18F]FPHCys are taken up by the L-transporter system. [18F]-L-FPHCys and [18F]-D-FPHCys displayed good stability
In Vivo without incorporation into protein at least 2 h postinjection. Biodistribution studies demonstrate good uptake in A375
tumor-bearing rodents with tumor to blood ratios of 3.5 and 5.0 for [18F]-L-FPHCys and [18F]-D-FPHCys, respectively, at 2 h
postinjection.
’ INTRODUCTION
lymphoma,7 lung,8 and breast cancer.9 Initial research and
applications focused on carbon-11-labeled amino acids10 and in
particular S-(2-[11C]methyl)-L-methionine ([11C]MET), which
has been extensively used in PET tumor imaging in both animals
and man.6
Metabolic imaging employing positron emission tomography
(PET) and [18F]fluoro-2-deoxy-D-glucose ([18F]FDG) has be-
come firmly established as a routine clinical tool in tumor staging
and for assessing tumor response to therapy. However, the high
uptake of this tracer in the brain and nonmalignant, inflammatory
tissue is a recognized limitation. New tracers are required, with
greater sensitivity in areas of high physiological [18F]FDG uptake
and better specificity for tumor, particularly in situations, includ-
ing surgery and irradiation, where inflammation is a part of the
normal healing response or may be a complication of cancer or
other diseases.1
It has long been recognized that amino acids play an important
role in the growth of tumor cells,2 providing an excellent
opportunity for their use in clinical cancer imaging. Furthermore,
the observation that amino acid imaging is less influenced by
inflammatory processes suggests that they may overcome some
of the disadvantages displayed by [18F]FDG.3 Several studies
have already confirmed the clinical value of radiolabeled amino
acids such as methionine (MET), fluoroethyl tyrosine (FET),
and 2-fluorodopa (FDOPA) for imaging tumors in the brain.4-6
There are less data relating to the utility of these agents in tumors
outside the brain, but there are some encouraging reports in
However, the short half-life of carbon-11 has prompted the
development of [18F]-labeled amino acids (t1/2 = 110 min) that
are more convenient for routine PET applications.11 Several
[18F]-labeled amino acids such as 2-[18F]fluoro-L-tyrosine
([18F]FT)12-14 and 4-[18F]fluoro-L-phenylalanine ([18F]FPhe),15
which are also incorporated into protein, have been evaluated
and potentially allow the study of protein synthesis rates.10,16
Although these amino acids have demonstrated encouraging
imaging characteristics, their radiosynthesis is tedious and results
in low radiochemical yield. Other [18F]-labeled amino acids such
as 2-[18F]fluoro-R-methyl-L-tyrosine ([18F]FMT),17,18 O-(2-[18F]-
fluoroethyl)-L-tyrosine ([18F]FET),6,19-21 and [18F]FDOPA are
not incorporated into protein but have demonstrated value in
studying amino acid transport processes in tumors.20
Received: November 24, 2010
Published: February 25, 2011
Published 2011 by the American Chemical Society
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Scheme 1. Synthesis of Precursors 1-3a
a Reagents and conditions: (a) Boc2O, dioxane, Na2CO3(aq), room temperature. (b) tert-Butyl-2,2,2-trichloroacetimidate, CH2Cl2, room temperature.
(c) Tributylphosphine, DMF, room temperature. (d) 1-Bromo-3-halopropane or 3-bromopropanol, K2CO3, DMF, room temperature. (e) p-Toluene-
sulfonyl chloride, N,N,N0,N0-tetramethyl-1,6-hexanediamine, acetonitrile, room temperature.
Although some of these [18F]-labeled amino acids have shown
higher sensitivity in the evaluation of brain tumors than
[18F]FDG, their applications outside of the brain have generally
been limited due to relative poor tumor uptake and substantial
uptake in normal tissues, particularly skeletal muscle, yielding
low to moderate tumor to background ratios. Recently, 2-amino-
3-[18F]fluoro-2-methylpropanoic acid ([18F]FAMP),22 3-
[18F]fluoro-2-methyl-2-(methylamino)propanoic acid ([18F]N-
MeFAMP),23 1-amino-3-[18F]fluorocyclobutyl-1-carboxylic acid
([18F]FACBC),24-26 and 1-amino-3-[18F]fluoromethyl-cyclo-
butane-1-carboxylic acid ([18F]FMACBC)27 were synthesized
with good tumor to brain ratios. Interestingly, the cyclobutyl
amino acids have also shown promising results in imaging
prostate tumors in man.
[11C]MET imaging. In addition, further selectivity and specificity
may be obtained by reducing the nonspecific signal by studying
the D enantiomer of the fluoroalkyl derivatives. Finally, the
incorporation of fluorine-18 onto a MET derivative would allow
wider clinical applications due to the comparative longer half-life
and wider distribution capability of this tracer to PET centers that
are remote from a cyclotron.
Recently, a new amino acid analogue of MET, S-(2-[18F]-
fluoroethyl)-L-homocysteine ([18F]FEHCys),30,31 was synthesized.
However, in our hands, [18F]FEHCys was found to be unstable
in aqueous systems, significantly limiting its use. For this reason,
we have developed the D and L enantiomers of the fluoropropyl
derivative S-(3-[18F]fluoropropyl)homocysteine ([18F]FPHCys).
Here, we describe the preparation of [18F]-L-FPHCys and [18F]-
D-FPHCys via a one-pot, two-step synthesis using the GE
Tracerlab FXFN synthesis module and report preliminary in vitro
and In Vivo pharmacological evaluation.
The convenient automated radiosynthesis of [11C]MET via a
[11C]methyl thio-methylation and its role in amino acid trans-
port and protein synthesis has made this amino acid one of the
most frequently used in tumor imaging studies. However, its
specificity and selectivity arre somewhat compromised due to the
presence of competing biochemical reactions and the formation
of a considerable amount of both protein and nonprotein
radiolabeled metabolites. First, MET plays a very significant
and competitive role in the activated methyl cycle, which serves
as a methyl group donor in many biosynthetic steps through the
formation of S-adenosylmethionine.3,28 Consequently, a consid-
erable amount of the carbon-11 activity coming from the
[11C]MET will contribute significantly to the background signal.
Second, the incorporation of [11C]MET into protein and the
subsequent degradation of these products generate a consider-
able amount of radiolabeled metabolites.29 Thus, the presence of
both protein and nonprotein metabolites significantly contri-
butes to a nonspecific signal, making quantitative imaging of
amino acid transport and protein synthesis rates in tumors
difficult.
’ RESULTS AND DISCUSSION
Chemistry. Our initial aim was to establish an efficient one-
pot synthesis of [18F]FPHCys using classical nucleophilic sub-
stitution reactions employing standard reagents and materials
with retention of stereochemistry and suitable for routine clinical
applications. Consequently, protected S-(3-tosyloxypropyl)homo-
cysteine 1, S-(3-bromopropyl)homocysteine 2, and S-(3-chloro-
propyl)homocysteine 3 were synthesized as precursors and
labeled with [18F]fluoride. Although the tosyloxy derivative is
generally the precursor of choice in [18F]radiofluorination reac-
tions, the corresponding brominated and chlorinated analogues
were also prepared due to their ease of synthesis, stability, and
good overall reactivity during radiolabeling and yield optimiza-
tion. The synthesis of these precursors was achieved in four (2
and 3) or five steps (1) (Scheme 1).
Therefore, the development of an S-alkyl MET derivative that
also incorporates a fluorine atom may first eliminate the compe-
titive activated “methyl cycle” reactions by preventing the
formation of S-adenosylmethionine and second reduce the
capacity for incorporation into protein, thereby limiting the
likelihood of metabolite production. Together, these effects
could significantly reduce the nonspecific signal inherent in
Briefly, reaction of L-homocystine or D-homocystine with di-
tert-butyl dicarbonate32,33 gave the protected compound 4, which
upon reaction with tert-butyl-2,2,2-trichloroacetimidate22,23,30 in
dichloromethane at room temperature afforded quantitatively
the disulfide 5. Cleavage of the disulfide bond to form protected
homocysteine 6 was achieved in 90% yield, using tributylpho-
sphine in dimethylformamide (DMF).30,33-35 Precursors 2 and
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Scheme 2. Radiosynthesis of [18F]FPHCys
Figure 1. Radiochromatograms showing the enantiomeric purity of [18F]-L-FPHCys using K2CO3 (A) or K2C2O4 (B) and [18F]-D-FPHCys using
K2C2O4 (C) during the radiolabeling. [18F]-L-FPHCys and [18F]-D-FPHCys have 10.5-11.5 and 13-14 min, respectively, as retention times.
3 and compounds 7 and 8 were prepared in 77-96% yields by
coupling 1-bromo-3-halopropane or 3-bromopropanol with the
thiol 6 in the presence of potassium carbonate in DMF at room
temperature.34,35 Protection of alcohol 8 with p-toluenesulfonyl
chloride in the presence of N,N,N0,N0-tetramethyl-1,6-hexane-
diamine in acetonitrile was achieved in 77% yield.36 The overall
yields for the preparation of the tosyl-, bromo-, and chloro-
precursors 1-3 were 65, 68, and 83%, respectively, for both the L
and the D-enantiomers. Using this synthetic scheme, the enan-
tiomeric purity of the final compounds as confirmed by chiral
high-pressure liquid chromatography (HPLC) analysis was
found to exceed 98%.
Radiochemistry. The initial radiosynthesis and yield optimi-
zation studies of [18F]FPHCys involved the reaction of precursor
1 using classical [18F]fluoride nucleophilic substitution22,23,37
reactions in the presence of K2.2.2. and potassium carbonate,
followed by acid hydrolysis of the protecting groups (Scheme 2).
Briefly, the L-isomer of precursor 1 (10-14 μmol) was allowed
to react with [18F]fluoride in acetonitrile containing 10 mg of
K2.2.2. and 2 mg of potassium carbonate at 100 °C for 15 min.
Attempts to increase the radiochemical yield by extending the
reaction time to 30 min led to a decreased yield of 73%, most
likely due to decomposition. The intermediate [18F]7 was
subsequently hydrolyzed with 6 N HCl at 100 °C for 5 min
followed by neutralization with 6 N NaOH and dilution in
phosphate buffer 1.5 M, pH 6. The resulting solution was purified
by HPLC, but to increase the HPLC separation efficiency of
[18F]-L-FPHCys, evaporation of the acetonitrile posthydrolysis
was required. Consequently, [18F]-L-FPHCys was synthesized
with an overall radiochemical yield of 30 ( 5% nondecay-
corrected and a radiochemical purity >98%.
However, chiral HPLC analysis indicated that under these
reaction conditions a partial racemization of the [18F]-L-FPHCys
had occurred, resulting in a final [18F]-L-FPHCys/[18F]-D-
FPHCys ratio of 75:25 (Figure 1). Further analysis of both
reaction steps indicated that racemization occurred during the
initial radiolabeling step, brought about by proton abstraction of
the R-hydrogen by the potassium carbonate base. Attempts to
prevent or reduce racemization by varying the reaction condi-
tions and precursor (2 and 3), for example, by decreasing
reaction time and temperature (even down to 50 °C), were
unsuccessful.
To avoid proton abstraction at the chiral center, one approach
was to use either more hindered protecting groups or an
alternative base. The use of N-trityl amino and O-tert-butyl ester
protecting groups, as employed in the radiosynthesis of FET,
successfully gave [18F]FET in a moderate radiochemical yield
with retention of chirality when potassium carbonate was em-
ployed as a base.37,38 Another simpler approach was to use a
weaker base with a pKa < 10 (such as tetrabutylammonium
hydrogencarbonate or potassium oxalate) for the labeling instead
of potassium carbonate. In this study, potassium oxalate was
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chosen because of the simplicity in reaction workup and purifica-
tion. Thus, the radiolabeling of [18F]-L-FPHCys was investigated
with potassium oxalate (2.6 mg), K2.2.2. (10 mg), and precursors
1-3 (5 mg, 10-14 μmol) in acetonitrile at 50, 80, and 100 °C
for 30 min. Under these conditions, the radiochemical yield
obtained for the synthesis of the intermediate [18F]7 increased
with temperature, with the highest at 100 °C for 30 min (62, 48,
and 24% for precursors 1, 2, and 3, respectively). In contrast to
the use of potassium carbonate, potassium oxalate gave lower
radiochemical yields for each precursor 1-3 investigated. Acid
hydrolysis of the intermediate [18F]7 (generated from 1 for 10
min at 100 °C in 57% yield) with 6 N HCl at 100 °C for 5 min,
followed by neutralization with 6 N NaOH, dilution with
phosphate buffer 1.5 M, pH 6, and purification by HPLC
produced [18F]-L-FPHCys with a radiochemical purity of >98%
and without racemization (Figure 1).
Figure 2. Cellular uptake of [18F]-L-FPHCys and [18F]-D-FPHCys in
A375 and HT29 tumor cells. Data are means ( SDs (n = 3).
Finally, [18F]-L-FPHCys was synthesized from precursor 1 by
a one-pot, two-step synthesis, which has been fully automated on
a GE Tracerlab FXFN synthesis module in a reproducible
nondecay-corrected radiochemical yield of 20 ( 5% and with a
specific activity >185 GBq/μmol in approximately 65 min of
preparation time including HPLC purification, formulation, and
sterile filtration through a 0.22 μm filter for biological studies.
The radiosynthesis of [18F]-D-FPHCys from the precursor 1-3
(D-isomer) was accomplished using the same process as for the
[18F]-L-FPHCys and gave the same results with an enantiomeric
purity >98% (Figure 1).
selective for the L-isomer over the D-isomer as the uptake of both
isomers was blocked by BCH. This observation is consistent with
that found for [11C]MET.39
System L is responsible for transporting large neutral amino
acids with branched or aromatic side chains such as leucine,
isoleucine, valine, phenylalanine, tyrosine, tryptophan, histidine,
and MET.40 A subtype of this transport system, the L type amino
acid transporter 1 (LAT1), has been reported to lack marked
stereoselectivity.40 The authors were able to demonstrate trans-
port of 14C-labeled D-leucine and D-phenylalanine by human
LAT1 with high affinity and also showed that human LAT1-
mediated transport of 14C-leucine was inhibited by D-leucine,
D-phenylalanine, and D-MET. Similarly, rat LAT2 transport of
14C-leucine is also inhibited by D-isomers of serine, cysteine, and
asparagines.41 Therefore, the results from the in vitro cell uptake
studies indicate that substitution of the S-methyl with an S-
fluoropropyl moeity on MET did not affect its amino acid
transport properties.
To investigate the biochemical properties of this new fluor-
opropyl MET derivative, cellular uptake studies, protein incor-
poration, metabolite, biodistribution, and imaging studies were
performed for each enantiomer.
In Vitro Evaluation of [18F]FPHCys. Cellular uptake studies
of both [18F]-L-FPHCys and [18F]-D-FPHCys were conducted
in A375 and HT29 tumor cell lines (Figure 2). In A375 cells,
[18F]-L-FPHCys rapidly accumulated with more than 50% of the
total radioactivity accumulation occurring within the first 2 min
of incubation. Uptake continued to increase for 60 min, reaching
a maximum of 9% per 105 cells. Uptake of [18F]-D-FPHCys was
significantly lower than [18F]-L-FPHCys at all time points. The
uptake kinetics of [18F]-D-FPHCys in A375 cells was rapid for the
first 15 min and continued to increase at a slower rate from 15 to
60 min with the highest (6.8% per 105 cells) observed at 60 min.
Cellular uptake in the HT29 cell line was significantly lower for
both enantiomers with a maximum value of 2 and 1.6% per 105
cells for [18F]-L-FPHCys and [18F]-D-FPHCys, respectively,
over 4 h.
In Vivo Biodistribution of [18F]FPHCys. The tissue distribu-
tion and kinetics of uptake for L- and D-enantiomers of
[18F]FPHCys in A375 tumor-bearing mice is summarized in
Table 1. As expected, the % ID/g of [18F]-L-FPHCys in all tissue
(except the pancreas for the first hour) and blood was higher than
that of [18F]-D-FPHCys throughout the study. Consequently,
the D-enantiomer exhibited a more rapid kidney extraction and
clearance into the bladder. This observation was consistent with
those reported for L- and D-[11C]MET, O-11C-methyl tyrosine,
and O-18F-fluoromethyl tyrosine.44 The liver displayed a signifi-
cantly higher uptake of [18F]-L-FPHCys than the corresponding
[18F]-D-FPHCys with both displaying rapid clearance over the
2 h period studied. At 2 h, the uptake of the D-enantiomer in the
liver was approximately one-third that of the L-enantiomer.
Interestingly, the uptake of [18F]-D-FPHCys in the pancreas
was higher than the corresponding L-enantiomer for the first 30
min, but its clearance rate was faster than the L-enantiomer,
resulting in a lower uptake from 1 h onward. Although the
absolute uptake of [18F]-L-FPHCys in the tumor was signifi-
cantly higher than the D-enantiomer over the 2 h time period, the
tumor-to-blood ratio of [18F]-D-FPHCys was significantly higher
than that of [18F]-L-FPHCys (Figure 4). Both tracers showed
longer retention in the tumor after 2 h as compared to the other
organs in the periphery. Uptake of activity in the bone was lower
for the D-enantiomer with both enantiomers displaying a steady
decline. Although the bone/marrow composition was not ana-
lyzed individually, the declining activity over the 2 h period is not
To characterize the amino acid transport pathway used for
[18F]FPHCys uptake in tumor cells, competitive inhibition
experiments were performed (Figure 3). Pretreating cells with
2-amino-bicyclo[2.2.1]heptane-2-carboxylic acid (BCH), an in-
hibitor of the amino acid transport system L, decreased the
uptake of [18F]-L-FPHCys by 98% in the A375 cell line and 86%
in HT29 cells when compared to controls (P < 0.05). Uptake of
[18F]-D-FPHCys was decreased by 94 and 89% in A375 and
HT29 cells, respectively (P < 0.05). In contrast, the A type amino
acid transport inhibitor; N-methyl-R-aminoisobutyric acid
(MeAIB) did not significantly block the uptake of [18F]-L-
FPHCys or [18F]-D-FPHCys in either cell line. Therefore,
cellular uptake of [18F]-L-FPHCys and [18F]-D-FPHCys in
tumor cells was determined to be primarily via the amino acid
transport system L. While uptake in A375 cells of [18F]-L-
FPHCys was higher than [18F]-D-FPHCys, this uptake was not
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Figure 3. Blocking effect of specific amino acid transport inhibitors on [18F]FPHCys uptake in A375 (A) and HT29 (B) cells. Data are means ( SDs
(n = 3).
Table 1. Tissue Distribution of [18F]-L-FPHCys and [18F]-D-
FPHCys in Balb/c Nude Mice Bearing A375 Tumorsa
organ isomer
15 min
30 min
60 min
120 min
liver
L
5.17 ( 0.74 4.84 ( 0.57 2.75 ( 0.43 1.92 ( 0.45
3.96 ( 0.67 3.05 ( 0.38 1.49 ( 0.16 0.16 ( 0.69
5.05 ( 0.95 4.18 ( 0.64 2.11 ( 0.38 1.39 ( 0.31
4.24 ( 0.79 2.83 ( 0.30 0.95 ( 0.14 0.31 ( 0.05
15.4 ( 1.20 12.6 ( 1.30 5.96 ( 0.61 3.92 ( 0.87
12.2 ( 1.66 6.49 ( 1.20 2.75 ( 0.22 1.04 ( 0.28
4.12 ( 0.59 4.20 ( 0.37 2.52 ( 0.31 1.52 ( 0.37
2.12 ( 0.40 1.97 ( 0.40 1.44 ( 0.05 0.63 ( 0.17
3.33 ( 0.58 3.50 ( 0.57 2.31 ( 0.26 1.69 ( 0.29
2.55 ( 0.41 2.29 ( 0.16 1.63 ( 0.10 1.05 ( 0.12
5.21 ( 0.32 5.07 ( 0.60 2.62 ( 0.33 1.63 ( 0.42
4.11 ( 0.75 2.84 ( 0.75 1.55 ( 0.22 0.85 ( 0.13
5.08 ( 0.36 4.20 ( 0.15 2.32 ( 0.46 1.37 ( 0.39
3.92 ( 0.76 3.05 ( 0.40 1.16 ( 0.08 0.40 ( 0.16
3.13 ( 0.58 2.57 ( 0.33 1.30 ( 0.25 2.57 ( 0.331
1.53 ( 0.15 1.56 ( 0.29 0.75 ( 0.04 0.25 ( 0.03
14.3 ( 2.66 12.3 ( 2.34 6.97 ( 1.18 3.77 ( 0.89
37.1 ( 7.30 19.6 ( 3.97 4.79 ( 0.81 1.43 ( 0.25
5.17 ( 0.80 4.44 ( 0.54 2.32 ( 0.33 1.38 ( 0.37
3.51 ( 0.68 2.51 ( 0.35 0.89 ( 0.10 0.89 ( 0.100.3
9.41 ( 1.21 8.28 ( 0.81 5.64 ( 0.95 4.40 ( 0.67
6.01 ( 0.77 5.87 ( 1.14 3.91 ( 0.32 1.68 ( 0.27
D
L
spleen
kidney
muscle
bone
D
L
D
L
D
L
Figure 4. Time course of tumor-to-blood ratios of [18F]-L-FPHCys and
[18F]-D-FPHCys in A375 tumor-bearing mice. The ratio of % ID/g of
tumor against blood was determined for each time point in the
biodistribution study.
D
L
lungs
D
L
heart
D
L
samples collected at 0.25 and 1 h. The radioactivity collected
from the 2 h samples was too low for detection by HPLC;
therefore, metabolites were only analyzed up to 1 h in these
tissues.
brain
D
L
pancreas
blood
tumor
From the extracted activity, HPLC analysis indicated that
more than 80% of the tracer remained intact after 2 h for both the
L- and the D-enantiomers of [18F]FPHCys in all tissues analyzed.
In plasma, the percentage of unchanged [18F]-L-FPHCys and
[18F]-D-FPHCys decreased from 90% at 0.25 h to 78% at 2 h
postinjection. The composition of radioactive material extracted
from the urine indicated that only 15% corresponded to intact
[18F]-L-FPHCys at 0.25 h postinjection, which was reduced to
less than 4% at 2 h. In contrast, more than 75% of radioactivity in
urine corresponded to intact [18F]-D-FPHCys at 2 h postinjec-
tion. In the pancreas, more than 95% of the radioactivity
represented [18F]-D-FPHCys at 0.25 and 1 h. However, for the
L-isomer, only 30% of radioactivity in the pancreas was intact
tracer at 1 h. For both isomers, more than 90% of unchanged
tracer was measured in the brain after 1 h.
A comparison of the metabolic stability of the two enantio-
mers of [18F]FPHCys revealed that the D-isomer was more stable
in all tissues studied, whereas the L-isomer was metabolized much
faster as gauged by analysis of radioactive material in the urine
and pancreas. Despite this, [18F]-L-FPHCys was still remarkably
stable in the plasma after 2 h. During the HPLC analysis, it was
D
L
D
L
D
a Means ( SDs (n = 5). Data are expressed as % injected dose/g tissue.
indicative of any significant defluorination and subsequent bone
fluoride uptake but most likely bone marrow uptake. This was
further supported by PET/CT images, indicating uptake in bone
regions with extensive marrow content (Figure 6).
Metabolite Studies. The stability and fate of [18F]-L-
FPHCys and [18F]-D-FPHCys over time was analyzed in tumor,
plasma, urine, pancreas, and brain (cortical) samples (Figure 5).
The amount of unchanged tracer in tissue samples was deter-
mined by radio-HPLC.42-44 After ultrasonic disruption of tissues
in saline and centrifugation, at least 82% of the radioactivity in the
tumor was recovered in the supernatant from samples collected
at 0.25, 1, and 2 h for HPLC analysis. At least 85% of the
radioactivity was recovered from the pancreas and brain from
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Figure 5. Percentage of unmetabolized [18F]-L-FPHCys (A) and [18F]-D-FPHCys (B) in plasma, tumor, brain (cortex), pancreas, and urine, as
measured using radio-HPLC. The percentage incorporation of [18F]-L-FPHCys (C) and [18F]-D-FPHCys (D) into proteins in plasma, tumor, cortex,
and pancreas, as measured using protein precipitation with trichloroacetic acid, was 0.25, 1, and 2 h after injection.
shown that the main radioactive metabolites in tissues were
highly polar products as they eluted with the solvent front. In a
recent study, the stability of the L- and D-isomers of the unnatural
amino acids, O-[11C]methyl tyrosine ([11C]CMT) and O-[18F]-
fluoromethyl tyrosine ([18F]-FMT), was compared to the L- and
D-isomer of [11C]MET.44 Metabolic analysis of the plasma
demonstrated rapid metabolism of the natural amino acid [11C]-
L-MET, whereas the unnatural D-isomer remained unmetabo-
lized up to 1 h. However, both isomers of the unnatural amino
acids, [11C]CMT and [18F]FMT, also remained stable in the
plasma. Finally, [18F]-L-FPHCys and [18F]-D-FPHCys were
shown to be sufficiently stable In Vivo, 2 h after iv injection, to
allow for their use as PET tracers of amino acid uptake in tumors.
Incorporation of [18F]-L-FPHCys and [18F]-D-FPHCys into
Proteins. The extent of protein incorporation of [18F]FPHCys
was determined by protein precipitation with trichloroacetic acid
(TCA)42,43,45,46 (Figure 5) and size exclusion chromatography
(SEC)42 at 0.25, 1, and 2 h postinjection. Quantification of the
incorporation of [18F]-L-FPHCys and [18F]-D-FPHCys into
proteins by protein precipitation on aqueous extracts of tumor,
frontal cortex, pancreas, and plasma homogenates resulted in
only 1-4% of radioactivity present in the protein fractions
(Figure 5). However, SEC of these samples showed no evidence
of [18F]-L-FPHCys and [18F]-D-FPHCys incorporation into
proteins even after 2 h due to the lower sensitivity of this
Figure 6. Coronal PET/CT images of Balb/c nude mice with A375
tumors (arrow) in the left flank at 60 min postinjection of [18F]-L-
FPHCys (A) and [18F]-D-FPHCys (B).
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technique. Unlike [11C]methyl-L-MET, which is actively incor-
porated into proteins, the structural modifications of the [18F]-L-
FPHCys and [18F]-D-FPHCys indicated no incorporation.
PET Imaging Studies. The whole body distribution of [18F]-
L-FPHCys and [18F]-D-FPHCys in A375 tumor-bearing mice
(n = 3) was followed over 120 min using PET/CT imaging.
Typical coronal PET images of both the [18F]-L-FPHCys and the
[18F]-D-FPHCys administered into A375 tumor-bearing mice
acquired at 60 min following radiotracer injection is shown in
Figure 6.47 In these images, the highest radioactivity concentra-
tion was observed in the kidneys, followed by the tumor. The
radioactivity observed in the bladders of mice injected with [18F]-
D-FPHCys was higher than those injected with [18F]-L-FPHCys.
A lower background activity [18F]-D-FPHCys in muscle and liver
suggests potential advantages for clinical imaging. These obser-
vations confirmed the quantitative results obtained in the bio-
distribution studies. Interestingly, significant tracer uptake was
observed in bone/marrow structures, confirming the biodistri-
bution studies.
deuterated solvent (CDCl3, D2O). Chemical shifts are reported as parts
per million (δ) relative to tetramethylsilane (TMS, 0.00 ppm), which
was used as an internal standard. Coupling constants are given in Hz, and
coupling patterns are abbreviated as follows: s, singlet; d, doublet; t,
triplet; q, quadruplet; qu, quintuplet; m, mutiplet; and dt, doublet of
triplet. Low-resolution mass spectrometry (LRMS) was performed on a
Waters Micromass ZQ Quadrupole Mass Spectrometer, whereas high-
resolution mass spectrometry (HRMS) was performed using a Micro-
mass Qtof Ultima or AutoSpec TOF. TLCs were run on precoated
aluminum plates of silica gel 60F254 (Merck), and Rf values were
established using an UV lamp at 254 nm or spraying with reagent.
Column chromatography was undertaken on Merck 60 silica gel (40-
63 μm) columns. The purity of all test compounds and key intermedi-
ates was determined to be >95% using an analytical HPLC, consisting of
a Waters 600 gradient pump, a Waters 2489 UV detector (λ = 210 and
254 nm), and a Phenomenex Bondclone C18 column (300 mm ꢀ 7.8
mm, 10 μm). HPLC was run at 2 mL/min with acetonitrile (eluent I)
and water (eluent II) and the following gradient: 1% acetonitrile for 3
min, ascending to 100% in 15 min and 100% for 10 min.
[18F]HF was produced on a GE PET trace Cyclotron via the 18O(p,
n)18F nuclear reaction (Cyclotek, Australia). The radiolabeling was
performed on a GE Tracerlab FXFN synthesis module (GE Medical
Systems, Milwaukee, WI). The intermediate product labeled with
fluorine-18 was analyzed by HPLC, consisting of a Waters 510 pump, a
Linear UVIS detector (λ = 210 nm) in series with a Berthhold βþ-flow
detector, on a Phenomenex Bondclone C18 column (300 mm ꢀ 7.8 mm,
10 μm) at 2 mL/min with CH3CN/H2O (75:25, v/v) as the mobile
phase. S-(3-[18F]fluoropropyl) homocysteine was purified by HPLC
available on GE TracerLab FXFN module, consisting of a Sykam S-1021
pump, a Knauer K-2001 UV detector (λ = 220 nm) in series with a
Berthhold βþ-flow detector, on a Waters Atlantis C18 T3 column (250
mm ꢀ 10 mm, 5 μm) at 3 mL/min with ethanol/phosphate buffer, pH 6,
0.15 M (5:95, v/v) as the mobile phase. Quality control analysis of
S-(3-[18F]fluoropropyl) homocysteine was performed on a Varian 9002
pump and a Linear UV-vis detector (λ = 226 nm) in series with an Ortec
ACE Mate βþ-flow detector on a Phenomenex Gemini column (150 mm
ꢀ 4.6mm, 5 μm) at1 mL/minwith phosphatebuffer, pH 11.5, 0.05 M, as
the mobile phase for chemical and radiochemical purity and on a
Phenomenex Chirex D Penicillamine column (150 mm ꢀ 4.6 mm, 5
μm) at 1 mL/min with isopropanol/CuSO4 aq, 2 nM (1:9, v/v), as the
mobile phase for enantiomeric purity. The identity of the labeled
compound was confirmed by coinjection with the authentic compound
on HPLC. The specific activity was determined by injection of an aliquot
of the final solution with known volume and radioactivity on a Phenom-
enex Gemini analytical column (150 mm ꢀ 4.6 mm, 5 μm). The area of
the UV absorbance peak measured at 226 nm corresponding to carrier
product was measured and compared to a standard curve relating mass to
UV absorbance. Only a specific activity below 185 GBq/μmol can be
measure accurately due to the low absorbance of FPHCys in the UV.
Radioactivity was measured with a Capintec R15C dose calibrator.
Metabolite studies were performed on a system consisting of a Waters
600 gradient pump, a Waters 486 UV detector (λ = 210 nm) in series
with a β-Ram IN/US for measurement of radioactivity, and a Waters
Atlantis C18 T3 column (150 mm ꢀ 4.6 mm, 5 μm) at 1 mL/min with
methanol/phosphate buffer, pH 6, 0.15 M (5:95, v/v), as the mobile
phase. The protein-bound activity was determined using a SEC on a
system consisting of a Waters 600 gradient pump and a Waters 2489 UV
detector (λ = 210 and 254 nm) in series with a Posi-Ram IN/US for
measurement of radioactivity, on a Phenomenex BioSep SEC S2000
column (300 mm ꢀ 7.8 mm) at 1 mL/min with phosphate buffer, pH 7,
50 mM, as the mobile phase.
’ CONCLUSION
A new chemically stable, [18F]-fluoropropyl derivative of
MET was conveniently prepared from precursor 1 in a non-
decay-corrected radiochemical yield of 20 ( 5%, in high radio-
chemical and enantiomeric purity and high specific activity for
evaluation in tumor uptake studies. Unlike the fluoroethyl
derivatives, [18F]-L-FPHCys and [18F]-D-FPHCys displayed
good chemical stability for biological evaluation.
In Vivo studies of [18F]-L-FPHCys and [18F]-D-FPHCys in
A375 tumor-bearing mice indicate a distribution profile not
unlike that of [11C]MET with high uptake in the tumor (9 and
6% ID/g at 15 min, respectively). Significantly, both the [18F]-L-
FPHCys and the [18F]-D-FPHCys enantiomers displayed spe-
cific transport into tumor cells via the L-amino acid transporter.
However, most importantly, the S-fluoropropyl fragment on
MET inhibited the incorporation of both [18F]-L-FPHCys and
[18F]-D-FPHCys into protein. It appears that the structural
modifications made to MET via the introduction of the fluor-
oalkyl group renders this compound incompatible with the cell's
protein synthesis machinery, yielding lower background signals
for higher target to nontarget ratios. The fluoropropyl moiety of
FPM may also avoid In Vivo S-methyl transfer biochemistry,
potentially reducing side reactions and further reduce nonspe-
cific signal. [18F]-D-FPHCys, in particular, shows promising
imaging characteristics with lower uptake and faster clearance
of activity from normal tissues, resulting in a higher tumor to
background ratio than for [18F]-L-FPHCys.
’ EXPERIMENTAL SECTION
General. All reagents and solvents were purchased from Lancaster,
Merck, or Sigma-Aldrich. All chemicals and solvents were used without
further purification. N,N0-Di(tert-butoxycarbonyl)homocystine 4, (tert-
butyl) N,N0-bis(tert-butoxycarbonyl)homocystinate 5, and tert-butyl
N-(tert-butoxycarbonyl)homocysteinate 6 were prepared as described
by Zhu et al. and Bourdier et al.30,33 Sep-Pak Light QMA and Sep-Pak
Light Alumina N were purchased from Waters. Millex GS 0.22 μm sterile
filters were supplied from Millipore.
All melting points were determined in open capillary tubes using a
SRS Optimet automated melting point system, MPA 100, and are
A375 human amelanotic melanoma cells and HT29 human colon
adenocarcinoma cells were purchased from American Type Culture
Collection. RPMI-1640 medium and L-glutamine were supplied from
1
uncorrected. H and 13C NMR spectra were measured on a Bruker
DPX 400, at 400 MHz (1H) and 100 MHz (13C), in an appropriate
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Journal of Medicinal Chemistry
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Sigma-Aldrich, and 10% fetal bovine serum was purchased from
Invitrogen.
94%) as a yellow oil. HPLC purity, 99%. 1H NMR (CDCl3, 400 MHz): δ
5.15-5.05 (m, 1H), 4.35-4.20 (m, 1H), 3.64 (t, J = 6.7 Hz, 2H), 2.67 (t,
J = 6.7 Hz, 2H), 2.60-2.50 (m, 2H), 2.15-2.05 (m, 1H), 2.02 (qu, J =
6.7 Hz, 2H), 1.95-1.80 (m, 1H), 1.47 (s, 9H), 1.44 (s, 9H). 13C NMR
(CDCl3, 100 MHz): δ 171.43, 155.48, 82.36, 79.97, 53.53, 43.54, 33.32,
32.25, 29.16, 28.46, 28.16, 28.03. LRMS: ES(þve) m/z 368 (M[35Cl] þ
1), 370 (M[37Cl] þ 1). HRMS: ES(þve) calcd for C16H30NO4SCl
(M[35Cl] þ 1), 368.1662; found, 368.1677.
All experimental procedures were performed in compliance with the
NHMRC Australian Code of Practice for the care and use of animals for
scientific purposes. Female Balb/c nude mice (5 weeks) were obtained
from the Animal Resource Centre (WA, Australia). Mice were housed
under constant environmental conditions and were allowed free access
to food and water throughout the experimental period. In Vivo studies
were performed in these mice bearing the A375 human amelanotic
melanoma tumor. A375 cells were resuspended in Ca2þ- and Mg2þ-free
PBS at 1 ꢀ 107 viable cells per mL of which 0.1 mL was injected subcuta-
neously in the left flank of 6 week old mice.
tert-Butyl N-(tert-butoxycarbonyl)-S-(3-tosyloxypropyl)-
homocysteinate (1). To a solution of 8 (2.85 g, 8.20 mmol) in
acetonitrile (115 mL) was added N,N,N0,N0-tetramethyl-1,6-hexanedia-
mine, (2.84 g, 16.14 mmol) followed by p-toluenesulfonyl chloride (2.39
g, 12.30 mmol), and the reaction mixture was stirred for 4 h at room
temperature under nitrogen. The reaction mixture was diluted with
water (340 mL) and extracted with ethyl acetate (3 ꢀ 340 mL). The
combined organic layers were washed with brine (340 mL) and dried
(MgSO4), and the solvents evaporated. The crude residue was purified
by chromatography on silica gel (heptanes/ethyl acetate 80:20) to give 1
(3.19 g, 77%) as a yellow oil. HPLC purity, 99%. 1H NMR (CDCl3, 400
MHz): δ 7.78 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 5.15-5.05
(m, 1H), 4.30-4.15 (m, 1H), 4.12 (t, J = 6.7 Hz, 2H), 2.53 (t, J = 6.7 Hz,
2H), 2.50-2.40 (m, 2H), 2.45 (s, 3H), 2.10-1.95 (m, 1H), 1.90 (qu, J =
6.7 Hz, 2H), 1.90-1.75 (m, 2H), 1.46 (s, 9H), 1.43 (s, 9H). 13C NMR
(CDCl3, 100 MHz): δ 171.37, 155.47, 144.97, 133.15, 130.03, 128.05,
82.37, 79.97, 68.88, 53.51, 33.19, 29.82, 28.45, 28.14, 27.99, 27.97, 21.78.
LRMS: ES(þve) m/z 526 (M þ Na). HRMS: ES(þve) calcd for
C23H37NO7S2 (M þ 1), 504.2090; found, 504.2105.
The mouse was anaesthetized via inhalant isoflurane (Forthane,
Abbott) in 200 mL/min oxygen during the imaging study. PET imaging
was performed on an Inveon multimodality PET/CT system (Siemens
Medical Solutions, Knoxville, United States).
tert-Butyl N-(tert-butoxycarbonyl)-S-(3-fluoropropyl)-
homocysteinate (7). To a solution of 6 (445 mg, 1.53 mmol) in
DMF (6.7 mL) was added 1-bromo-3-fluoropropane (326 mg, 2.29
mmol) followed by potassium carbonate (422 mg, 3.05 mmol), and the
resultant white solution was stirred for 24 h at room temperature under
nitrogen. The reaction mixture was diluted with water (90 mL) and
extracted with ethyl acetate (3 ꢀ 45 mL). The combined organic layers
were washed with water (90 mL) and dried (MgSO4), and the solvents
evaporated. The crude residue was purified by chromatography on silica
gel (heptanes/ethyl acetate 90:10) to give 7 (504 mg, 95%) as a yellow
oil. HPLC purity, 98%. 1H NMR (CDCl3, 400 MHz): δ 5.15-5.05 (m,
1H), 4.53 (dt, J = 47.1 Hz, J = 5.8 Hz, 2H), 4.35-4.20 (m, 1H), 2.64 (t,
J = 7.3 Hz, 2H), 2.60-2.50 (m, 2H), 2.15-2.03 (m, 1H), 2.03-1.80 (m,
3H), 1.47 (s, 9H), 1.44 (s, 9H). 13C NMR (CDCl3, 100 MHz): δ 171.45,
155.48, 82.44 (J = 165.6 Hz), 82.35, 79.95, 53.55, 33.28, 30.54 (J = 20.7
Hz), 28.45, 28.15, 28.08, 27.90 (J = 5.4 Hz). LRMS: ES(þve) m/z 374
(M þ Na). HRMS: ES(þve) calcd for C16H30NO4SF (M þ 1), 352.1958;
found, 352.1970.
tert-Butyl N-(tert-butoxycarbonyl)-S-(3-hydroxypropyl)-
homocysteinate (8). This compound was synthesized as described
for 7 above, from 6 (6 g, 20.61 mmol) and 3-bromopropanol (4.43 mg,
30.92 mmol). The crude residue was purified by chromatography on
silica gel (heptanes/ethyl acetate 70:30) to give 8 (6.91 mg, 96%) as a
yellow oil. HPLC purity, 98%. 1H NMR (CDCl3, 400 MHz): δ 5.12-
5.05 (m, 1H), 4.35-4.20 (m, 1H), 3.80-3.70 (m, 2H), 2.64 (t, J = 6.7
Hz, 2H), 2.60-2.50 (m, 2H), 2.15-2.05 (m, 1H), 1.95-1.80 (m, 2H),
1.83 (qu, J = 6.7 Hz, 2H), 1.47 (s, 9H), 1.44 (s, 9H). 13C NMR (CDCl3,
100 MHz): δ 172.56, 156.56, 82.35, 81.06, 61.62, 54.51, 33.18, 31.98,
28.80, 28.47, 28.16, 28.02. LRMS: ES(þve) m/z 372 (M þ Na). HRMS:
ES(þve) calcd for C16H31NO5S (M þ 1), 350.2001; found, 350.2017.
tert-Butyl S-(3-bromopropyl)-N-(tert-butoxycarbonyl)-
homocysteinate (2). This compound was synthesized as described
for 7 above, from 6 (200 mg, 0.68 mmol) and 1,3-dibromopropane (420
mg, 2.06 mmol). The crude residue was purified by chromatography on
silica gel (heptanes/ethyl acetate 90:10) to give 2 (215 mg, 77%) as a
yellow oil. HPLC purity, 99%. 1H NMR (CDCl3, 400 MHz): δ 5.15-
5.05 (m, 1H), 4.35-4.20 (m, 1H), 3.51 (t, J = 6.7 Hz, 2H), 2.67 (t, J = 6.7
Hz, 2H), 2.60-2.50 (m, 2H), 2.15-2.05 (m, 1H), 2.10-2.05 (qu, J =
6.7 Hz, 2H), 1.95-1.80 (m, 1H), 1.47 (s, 9H), 1.44 (s, 9H). 13C NMR
(CDCl3, 100 MHz): δ 171.43, 155.47, 82.37, 79.98, 53.53, 33.34, 32.31,
32.21, 30.36, 28.47, 28.16, 28.02. LRMS: ES(þve) m/z 412 (M[79Br] þ
1), 414 (M[81Br] þ 1). HRMS: ES(þve) calcd for C15H28NO4SBr
(M[79Br]), 412.1157; found, 412.1165.
S-(3-Fluoropropyl)homocysteine Hydrochloride (FPH-
Cys). To a solution of the protected amino acid 7 (63 mg) in THF
(530 μL) was added 6 N hydrochloric acid (1.07 mL), and the reaction
mixture was stirred at room temperature for 3 h. The solvent was
removed under reduced pressure, and the resulting solid was washed
with ethyl acetate (3 ꢀ 7 mL) and dried under vacuum to give FPHCys
(38 mg, 91%) as a white solid; mp 107-109 °C. HPLC purity, 98%. 1H
NMR (D2O, 400 MHz): δ 4.64 (dt, J = 47.1 Hz, J = 5.8 Hz, 2H), 4.17 (t,
J = 6.2 Hz, 1H), 2.77 (t, J = 7.3 Hz, 2H), 2.75 (t, J = 7.0 Hz, 2H), 2.35-
2.15 (m, 2H), 2.10-1.95 (m, 2H). 13C NMR (D2O, 100 MHz): δ
173.52, 84.88 (d, J = 158.7 Hz), 53.52, 30.81, 30.69 (d, J = 19.9 Hz),
27.84 (d, J = 5.4 Hz), 27.64. LRMS: ES(þve) m/z 196 (M þ 1). HRMS:
ES(þve) calcd for C7H14NO2SF (M þ 1), 196.0808; found, 196.0814.
Radiosynthesis of [18F]FPHCys. The radiolabeling of [18F]-L-
FPHCys and [18F]-D-FPHCys was performed on a GE Tracerlab FXFN
synthesis module using an in-house reaction sequence. On the GE
Tracerlab FXFN synthesis module, aqueous [18F]fluoride solution (18-
37 GBq) was loaded on a QMA cartridge first activated with potassium
carbonate (10 mL, 0.10 M). The concentrated [18F]fluoride was eluted
into the reactor with 1 mL of a solution of K2C2O4/K2CO3 (2.55 mg/50
μg) and K2.2.2. (10 mg) in a 1:4 ratio of acetonitrile/water. The solvent
was evaporated under vacuum and a stream of helium at 70 °C for 7 min
and then at 120 °C under vacuum only for 5 min. To the activated
K2.2.2./potassium [18F]fluoride was added the tosylate precursor 1 (5
mg, 10 μmol) in acetonitrile (2 mL), and the mixture was heated at
100 °C for 10 min. The reactor was cooled to 30 °C, and 6 N HCl (500
μL) was added. After 3 min at 100 °C, the acetonitrile was evaporated
under a stream of helium at 100 °C for 2 min. The reactor was then
cooled to 30 °C and sequentially treated with 6 N NaOH (500 μL) and
phosphate buffer, pH 6 (1 mL, 1.5 M). This solution was directly passed
through a Sep-Pak Light Alumina N before purification on a preparative
reverse phase HPLC column (Atlantis C18 T3 column (250 mm ꢀ 10
mm, 5 μm); mobile phase ethanol/phosphate buffer, pH 6, 0.15 M
(5:95, v/v); flow rate, 3 mL/min; λ = 220 nm). The fraction containing
the labeled product [18F]FPHCys was collected between 15 and 16 min,
passed through a 0.22 μm filter, and recovered in a sterile vial for dilution
with sodium chloride. Routinely, [18F]FPHCys was ready for injection
tert-Butyl N-(tert-butoxycarbonyl)-S-(3-chloropropyl)-
homocysteinate (3). This compound was synthesized as described
for 7 above, from 6 (200 mg, 0.68 mmol) and 1-bromo-3-chloroethane
(327 mg, 2.06 mmol). The crude residue was purified by chromatog-
raphy on silica gel (heptanes/ethyl acetate 90:10) to give 3 (236 mg,
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in approximately 65 min. Typically, 4.1-7.8 GBq of [18F]-L-FPHCys or
[18F]-D-FPHCys was isolated in 20 ( 5% nondecay-corrected.
The chemical and radiochemical purity of [18F]-L-FPHCys and [18F]-
D-FPHCys were analyzed on a Phenomenex Gemini column (150 mm
ꢀ 4.6 mm, 5 μm) with a flow of 1 mL/min and phosphate buffer,
pH 11.5, 0.05 M, as the mobile phase. The retention time was between
13.5 and 14.5 min for [18F]-L-FPHCys and [18F]-D-FPHCys, and the
identity of the labeled compound was confirmed by coinjection with the
authentic [19F]FPHCys standard on HPLC. Typically, both enantiomers
of [18F]FPHCys were synthesized with a radiochemical purity >98%
and a specific activity >185 GBq/μmol starting from 18 to 37 GBq of
[18F]fluoride.
radio-HPLC analysis, at 0.25, 1, and 2 h after injection of 18-20 MBq of
[18F]-L-FPHCys or [18F]-D-FPHCys. Plasma (50 μL) and urine (50 μL)
were mixed with L-FPHCys or D-FPHCys (5 nmol in 5 μL of water) and
KF (5 nmol in 5 μL of water) for direct injection onto the HPLC. Tissue
samples (50 mg) in 500 μL of saline containing unlabeled FPHCys
compound and KF were exposed for 2 min to an ultrasonic probe, and
the mixture was centrifuged at 6000g for 5 min. The supernatant was
collected and directly injected in HPLC. The radioactivity of the
precipitate was measured to quantify the saline extraction efficiency.
Radio-HPLC analysis was performed using a reverse phase HPLC
column (Atlantis C18 T3 column (150 mm ꢀ 4.6 mm, 5 μm); mobile
phase methanol/phosphate buffer, pH 6, 0.15 M (5:95, v/v); flow rate, 1
mL/min; λ = 210 nm). The radioactivity peak corresponding to the
authentic FPHCys was compared to the total activity registered in the
radiochromatogram to give the fraction of unchanged ligand in the
sample.
The enatiomeric purity of [18F]-L-FPHCys and [18F]-D-FPHCys was
analyzed using chiral HPLC analysis, using a Phenomenex Chirex D
Penicillamine column (150 mm ꢀ 4.6 mm, 5 μm) with a flow of 1 mL/
min and isopropanol/CuSO4aq, 2 nM (1:9, v/v), as the mobile phase.
The retention time was 10.5-11.5 min for [18F]-L-FPHCys and 13-14
min for [18F]-D-FPHCys. The identity of each enantiomer was con-
firmed by coinjection with the L-FPHCys and D-FPHCys standard on
HPLC. Typically, [18F]-L-FPHCys and [18F]-D-FPHCys were synthe-
sized with an enantiomeric purity greater of >98%.
Cell Cultures. A375 human amelanotic melanoma cells and HT29
human colon adenocarcinoma cells lines were cultured in RPMI-1640
medium supplemented with 10% fetal bovine serum and 2 mM
L-glutamine. Cells were maintained in 175 cm2 flasks in a 5% CO2,
37 °C humidified incubator.
Incorporation of [18F]-L-FPHCys and [18F]-D-FPHCys into
Proteins. To determine the extent of protein incorporation of the two
different 18F-labeled MET analogues, the protein-bound activity was
determined by protein precipitation with TCA and SEC at 0.25, 1, and
2 h after injectionof 18-20 MBq of [18F]-L-FPHCys or [18F]-D-FPHCys.
Plasma (50 μL) and tissue samples (tumor, cortex, and pancreas)
(50 mg) in 500 μL of 10% TCA containing unlabeled L-FPHCys or
D-FPHCys (5 nmol in 5 μL of water) and KF (5 nmol in 5 μL of water)
were exposed for 2 min to an ultrasonic probe, and the mixture was
centrifuged at 6000g for 5 min. The supernatant was collected, and this
process was repeated twice on the remaining precipitate with 250 μL of
10% TCA. The radioactivity of the precipitate and supernatant was
measured to quantify the incorporation of [18F]FPHCys into proteins,
in the sample.
Additionally, plasma (50 μL) was mixed with L-FPHCys or D-
FPHCys (5 nmol) and KF (5 nmol) for direct injection onto SEC.
Tissue samples (50 mg) in 500 μL of saline containing unlabeled
FPHCys compound and KF were exposed for 2 min to an ultrasonic
probe, and the mixture was centrifuged at 6000g for 5 min. The
supernatant was collected and directly injected onto SEC. The radio-
activity of the precipitate was measured to quantify the saline extraction
efficiency. SEC analysis was performed using a size exclusion column
(Phenomenex BioSep SEC S2000 column (300 mm ꢀ 7.8 mm); mobile
phase phosphate buffer, pH 7, 50 mM; flow rate, 1 mL/min; λ = 210 and
254 nm). The radioactivity peak corresponding to the authentic
FPHCys was compared to the total activity registered in the radio-
chromatogram to give the fraction of quantify the incorporation of
[18F]FPHCys into proteins, in the sample.
Cell Uptake Studies. In 24-well culture plates, 2.5 ꢀ 105 cells
were seeded in complete medium and left to attach overnight. The
following day, the number of cells per well were counted in triplicate for
each cell line. Before incubation with the radiotracer, the growth media
were removed, and the cells were washed once with warm PBS to
remove all traces of growth media. [18F]-L-FPHCys and [18F]-D-
FPHCys were formulated in warm PBS. Freshly prepared radiotracer
(0.37 MBq in 500 μL) was added, and the cells were incubated at 37 °C
for 2, 15, 30, 60, 120, 180, and 240 min. Uptake was terminated by
removing the tracer solution and washing cells with ice-cold PBS.
Subsequently, the cells were lysed with 500 μL of 0.2 N NaOH.
Radioactivity in the cells was measured with a γ-counter. The results
were expressed as percent of applied dose per 1 ꢀ 105 cells. All activities
were corrected for decay.
Competitive Inhibition Studies in Cells. Inhibitor studies
were carried out to investigate the transport mechanism involved in
the uptake of [18F]-FPHCys in tumor cells. BCH was used for system L,
and N-methyl-R-aminoisobutyric acid (MeAIB) for system A. Cells
were prepared as described for uptake studies. Cells were preincubated
with 400 μL of inhibitor (12.5 mmol/L in PBS) or PBS (for controls) at
37 °C. Freshly prepared radiotracer (0.37 MBq in 100 μL) was added,
and the cells were incubated at 37 °C for 30 min. Uptake was terminated
as described for uptake studies. The percentage of uptake in the
treatment groups relative to the control group was determined.
Biodistribution Studies in Tumor-Bearing Mice. Biodistri-
bution studies were performed 26 days after tumor induction. The tissue
distribution of radioactivity was determined in A375 tumor-bearing mice
after intravenous injection of 0.8-1 MBq of [18F]-L-FPHCys or [18F]-D-
FPHCys in 100 μL of saline. At 15, 30, 60, and 120 min postinjection of
the radiotracer, groups of mice (n = 5) were sacrificed by CO2
administration followed by cervical fracture and dissected. Selected
organs were weighed, and the radioactivity was measured using a
γ-counter. The percent injected dose (% ID) was calculated by compari-
son with suitable dilutions of the injected dose. Radioactivity concentra-
tions were expressed as percent of injected dose per gram of wet tissue
(% ID/g).
PET Imaging Studies in Tumor-Bearing Mice. The mouse
was anaesthetized via inhalant isoflurane in 200 mL/min oxygen during
the imaging study. A 10 min CT scan was performed on each subject for
anatomical information, followed by a 2 h PET scan. The mouse was
injected intravenously with 18-20 MBq of [18F]-L-FPHCys or [18F]-D-
FPHCys in 100 μL of saline. Image acquisition commenced simulta-
neously with radiotracer injection. The data were histogrammed into 25
consecutive frames and reconstructed with an iterative algorithm (3D
OSEM/FastMap). PET and CT volumes are automatically coregistered
and visualized with Brainvisa (http://brainvisa.info).
’ ASSOCIATED CONTENT
S
Supporting Information. Reactivity of precursor 1-3
b
using potassium carbonate or potassium oxalate for the radio-
labeling, HPLC profile of [18F]FPHCys for the radiosynthesis
and QCanalysis, and reaction sequenceand preparation of reagent
for the [18F]FPHCys radiosynthesis. This material is available free
of charge via the Internet at http://pubs.acs.org.
Metabolite Studies. The determination of unchanged radiotracer
in the plasma, tumor, cortex, pancreas, and urine was performed by
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Journal of Medicinal Chemistry
ARTICLE
’ AUTHOR INFORMATION
ꢁ (10) Vaalburg, W.; Coenen, H. H.; Crouzel, C.; Elsinga, PH. H.;
Langstrom, B.; Lemaire, C.; Meyer, G. J. Amino acids for the measurement
of protein syntheis In Vivo by PET. Nucl. Med. Biol. 1992, 19, 227–237.
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W. J. G. Fluorinated amino acids for tumour imaging with positron
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L-[2-18F]fluorotyrosine, a new PET tracer of protein synthesis. J. Nucl.
Med. 1989, 30, 1367–1372.
Corresponding Author
*Tel: þ61 2 9717 3292. Fax: þ61 2 9717 9262. E-mail:
bts@ansto.gov.au.
’ ACKNOWLEDGMENT
We thank Allira Schuster-Woodhouse, Cathy D. Jiang, Dr.
Tien Pham, and Naomi Wyatt for their assistance with radio-
labeling development and metabolite studies and Drs. Oliver
Neels and Peter Roselt for their support in translating and
validating the synthetic process at the Peter MacCallum Cancer
Centre for ongoing characterization studies. This study was
funded by the Cooperative Research Centre for Biomedical
Imaging Development Ltd (CRC-BID), Bundoora, Victoria,
Australia, established and supported under the Australian Gov-
ernment's Cooperative Research Centres program.
(13) Langen, K.-J.; Jarosch, M.; M€uhlensiepen, H.; Hamacher, K.;
Br€oer, S.; Jansen, P.; Zilles, K.; Coenen, H. H. Comparison of fluor-
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’ ABBREVIATIONS USED
BCH, 2-amino-bicyclo[2.2.1]heptane-2-carboxylic acid; DMF,
dimethylformamide; HPLC, high-pressure liquid chromatogra-
phy; ID, injected dose; K2.2.2., 4,7,13,21,24-hexaoxa-1,10-diaza-
bicyco[8.8.8]hexacosane; OSEM, ordered subset expectation
maximization; PET, positron emission tomography; SEC, size
exclusion chromatography; TCA, trichloroacetic acid
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