Radiolabeled amino acids for tumor imaging with PET: Radiosynthesis and biological evaluation of 2-amino-3-[18F]fluoro-2-methylpropanoic acid and 3-[18F]fluoro-2-methyl-2-(methylamino)propanoic acid

Article abstract of DOI:10.1021/jm010241x

Novel radiopharmaceuticals, including amino acids, that target neoplasms through their altered metabolic states have shown promising results in preclinical and clinical studies. Two fluorinated analogues of α-aminoisobutyric acid, 2-amino-3-fluoro-2-methy

Full text of DOI:10.1021/jm010241x

2240
J . Med. Chem. 2002, 45, 2240-2249
Ra d iola beled Am in o Acid s for Tu m or Im a gin g w ith P ET: Ra d iosyn th esis a n d
Biologica l Eva lu a tion of 2-Am in o-3-[18F ]flu or o-2-m eth ylp r op a n oic Acid a n d
3-[18F ]F lu or o-2-m eth yl-2-(m eth yla m in o)p r op a n oic Acid
J onathan McConathy,† Laurent Martarello,† Eugene J . Malveaux,† Vernon M. Camp,† Nicholas E. Simpson,†
Chiab P. Simpson,† Geoffrey D. Bowers,‡ J effrey J . Olson,‡ and Mark M. Goodman*,†
Department of Radiology and Department of Neurosurgery at Emory University Hospital, School of Medicine,
1364 Clifton Road Northeast, Atlanta, Georgia 30322
Received May 31, 2001
Novel radiopharmaceuticals, including amino acids, that target neoplasms through their altered
metabolic states have shown promising results in preclinical and clinical studies. Two
fluorinated analogues of R-aminoisobutyric acid, 2-amino-3-fluoro-2-methylpropanoic acid
(FAMP) and 3-fluoro-2-methyl-2-(methylamino)propanoic acid (N-MeFAMP), have been radio-
labeled with fluorine-18, characterized in amino acid uptake assays, and evaluated in vivo in
normal rats and a rodent tumor model. The key steps in the syntheses of both radiotracers
involved the preparation of cyclic sulfamidate precursors. Radiosyntheses of both [18F]FAMP
and [18F]N-MeFAMP via no-carrier-added nucleophilic substitution provided high yields (>78%
decay-corrected) in high radiochemical purity (>99%). Amino acid transport assays using 9L
gliosarcoma cells demonstrated that both compounds are substrates for the A type amino acid
transport system, with [18F]N-MeFAMP showing higher specificity than [18F]FAMP for A type
transport. Tissue distribution studies in normal Fischer rats and Fischer rats implanted
intracranially with 9L gliosarcoma tumor cells were also performed. At 60 min postinjection,
the tumor vs normal brain ratio of radioactivity was 36:1 in animals receiving [18F]FAMP and
104:1 in animals receiving [18F]N-MeFAMP. On the basis of these studies, both [18F]FAMP
and [18F]N-MeFAMP are promising imaging agents for the detection of intracranial neoplasms
via positron emission tomography.
In tr od u ction
MRI, or [18F]FDG, allowing better planning of treat-
ment.7,8 Additionally, some studies suggest that the
degree of amino acid uptake correlates with tumor
grade, which could provide important prognostic informa-
tion.8-10
The development of radiolabeled amino acids for use
as metabolic tracers to image tumors using positron
emission tomography (PET) and single photon emission
computed tomography (SPECT) has been underway for
two decades. Although radiolabeled amino acids have
been applied to a variety of tumor types, their applica-
tion to intracranial tumors has received considerable
attention due to potential advantages over other imag-
ing modalities. After surgical resection and/or radio-
therapy of brain tumors, conventional imaging methods
such as computed tomography (CT) and magnetic
resonance imaging (MRI) do not reliably distinguish
residual or recurring tumor from tissue injury due to
the intervention and are not optimal for monitoring the
effectiveness of treatment or detecting tumor recur-
rence.1,2 The leading PET agent for diagnosis and
imaging of neoplasms, 2-[18F]fluorodeoxyglucose (FDG),
also has limitations in the imaging of brain tumors.
Normal brain cortical tissue shows high [18F]FDG
uptake as does inflammatory tissue, which can occur
after radiation or surgical therapy; these factors can
complicate the interpretation of images acquired with
[18F]FDG.3-6 A number of reports indicate that PET and
SPECT imaging with radiolabeled amino acids better
define tumor boundaries within normal brain than CT,
A number of amino acids, including [11C]R-ami-
noisobutyric acid (AIB), L-[11C]methionine (Met), L-[18F]-
fluoro-R-methyl tyrosine, O-(2-[18F]fluoroethyl) tyrosine,
and anti-1-amino-3-[18F]fluorocyclobutyl-1-carboxylic acid
(FACBC), have been successfully used for PET tumor
imaging in humans.8,11-14 AIB is a nonmetabolized R,R-
dialkyl amino acid that is actively transported into cells
primarily via the A type amino acid transport system.
System A amino acid transport is increased during cell
growth and division and has also been shown to be
upregulated in tumor cells.15-17 Studies of experimen-
tally induced tumors in animals and spontaneously
occurring tumors in humans have shown increased
uptake of radiolabeled AIB in the tumors relative to
normal tissue.11,18-21 The N-methyl analogue of AIB,
N-MeAIB, shows even more selectivity for the A type
amino acid transport system than AIB.22,23 N-MeAIB
has been radiolabeled with carbon-11 and is metaboli-
cally stable in humans.24
We have developed fluorinated analogues of AIB
suitable for labeling with 18F and use in PET imaging.
These agents are expected to demonstrate metabolic
stability in vivo due to their R,R-dialkyl branching and
to have the potential for remote distribution due to the
110 min half-life of 18F vs 20 min for 11C. We report here
the synthesis, radiolabeling, and biological evaluation
* To whom correspondence should be addressed. Tel.: (404)727-
9366. Fax: (404)727-4366. E-mail: mgoodma@emory.edu.
† Department of Radiology.
‡ Department of Neurosurgery.
10.1021/jm010241x CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/26/2002

Amino Acids for Tumor Imaging with PET
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2241
Sch em e 1. Synthesis of Fluorine-19 Amino Acids
FAMP (5a ) and N-MeFAMP (5b)a
Sch em e 2. Synthesis of 3-Benzyloxy-2-[N-(tert-
butoxycarbonyl)amino]-2-methylpropanoic Acid
tert-Butyl Ester (8)a
a
a
Reagents: (a) KCN, NH4Cl, H2O. (b) HCl and then (Boc)2O.
Reagents: (a) (NH4)2CO3, KCN, NH4Cl, 1:1 EtOH:H2O. (b) 5
(c) Aqueous HCl. (d) Cl3CC(dNH)OtBu, CH2Cl2. (e) NaH, DMF,
N NaOH, 180 °C and then (Boc)2O, 9:1 CH3OH:Et3N. (c)
CH3I.
Cl3CC(dNH)OtBu, CH2Cl2.
Sch em e 3. Synthesis of N-Substituted Amino Alcohols
12a ,ba
of 2-amino-3-fluoro-2-methylpropanoic acid (FAMP, 5a )
and 3-fluoro-2-methyl-2-(methylamino)propanoic acid
(N-MeFAMP, 5b), fluorinated analogues of AIB and
N-methyl AIB, respectively. The dominant mechanism
of cellular uptake of these radiotracers by 9L gliosar-
coma cells was determined in vitro using inhibitors of
amino acid transport. Tissue distribution studies in
normal and 9L gliosarcoma tumor-bearing rats were
carried out after intravenous administration of [18F]5a
and [18F]5b, and the tumor uptake of radioactivity was
compared to uptake in normal brain for both com-
pounds.
a
Reagents: (a) 10% Pd/C, H2, CH3OH. (b) pTsOH‚H2O, EtOH,
40 °C. (c) DMB-Cl, Et3N, CH2Cl2. (d) NaH, DMF, CH3I. pTsOH )
p-toluenesulfonic acid, and DMB ) bis(4-methoxyphenyl)methyl.
Resu lts a n d Discu ssion
for the synthesis of [18F]5b, which contains a secondary
amine, in the case of [18F]5a , it was necessary to utilize
an amino substituent that was suitable for cyclic sul-
famidate formation but could be readily removed during
radiosynthesis (see below). For both radiotracers, the
key steps in the preparation of the precursors for
radiolabeling involved the synthesis of secondary amino
alcohols, which could be converted to cyclic sulfami-
dates.
Ch em istr y. The syntheses of nonradioactive 5a and
5b are shown in Scheme 1. For 5a , the aminonitrile 1
was prepared from fluoroacetone using a Strecker type
reaction. To facilitate purification, the carbamate 2 was
prepared by treating the crude product of the acid
hydrolysis of 1 with di-tert-butyl dicarbonate followed
by flash chromatography. The amino acid 5a was
obtained as its salt in analytically pure form by treating
2 with aqueous HCl. Compound 2 could also be obtained
by treating 14a (see Scheme 4 for structure) with
tetrabutylammonium fluoride and derivatizing the crude
amino acid using di-tert-butyl dicarbonate. Preparation
of 5b was performed starting with the carbamate 2.
Treatment of 2 with tert-butyl-2,2,2-trichloroacetami-
date under neutral conditions25 provided the N-tert-
butoxycarbonyl (N-Boc) ester 3, which was alkylated
with methyl iodide and sodium hydride in dimethylfor-
mamide (DMF) to yield 4. Deprotection of 4 in aqueous
HCl provided amino acid 5b as its salt. Although
synthesis of 5a ,b via the aminonitrile intermediate was
straightforward, this strategy was not amenable to
radiosynthesis of [18F]5a and [18F]5b.
Initial attempts to prepare [18F]5a from a methane-
sulfonyl ester precursor failed due to lack of 18F incor-
poration into the molecule, presumably because of the
low reactivity of the â-carbon due to its neopentyl
character. As an alternative, cyclic sulfamidates were
attractive precursors because they have been used to
prepare a number of 18F radioligands and nonradio-
active R,R-disubstituted amino acid derivatives includ-
ing 3-fluoro-2-(4-methoxybenzylamino)-2-methylpro-
panoic acid methyl ester.26-29 However, there are cur-
rently noliterature reports ofcyclicsulfamidate formation
from primary amines. While this did not pose a problem
The R-methyl serine derivative 8 served as a common
intermediate in the syntheses of [18F]5a and [18F]5b and
was prepared as shown in Scheme 2. Treatment of
3-benzyloxypropanone with a buffered ammonium car-
bonate and potassium cyanide solution led to the
formation of the hydantoin 6. Alkaline hydrolysis of the
hydantoin followed by treatment of the crude amino acid
with di-tert-butyl dicarbonate gave the N-Boc acid 7. The
tert-butyl ester 8 was prepared from 7 using tert-butyl-
2,2,2-trichloracetimidate under neutral conditions.25
The synthesis of the amino alcohols 12a and 12b is
depicted in Scheme 3. To prepare 12a , the alcohol 9 was
obtained from the catalytic hydrogenolysis of the benzyl
ether 8. The bis(4-methoxyphenyl)methyl group, also
known as 4,4′-dimethoxybenzhydryl (DMB), was incor-
porated because it provided a secondary amine for cyclic
sulfamidate formation but could be rapidly removed
under acidic conditions.30 This arrangement permitted
N-dealkylation, hydrolysis of the sulfamate obtained
from nucleophilic ring opening, and hydrolysis of the
tert-butyl ester in a single step after incorporation of
18F. Selective removal of the Boc protecting group of 9
in the presence of the tert-butyl ester was achieved with
p-toluenesulfonic acid by modifying the procedure re-
ported by Goodacre et al.31 While the reaction did not

2242 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
McConathy et al.
Sch em e 4. Synthesis of Cyclic Sulfamidates 14a ,b and
Radiosynthesis of [18F]FAMP (5a ) and [18F]N-MeFAMP
(5b)a
F igu r e 1. Inhibition of uptake of [18F]FAMP (5a ) and [18F]-
N-MeFAMP (5b) by BCH and N-MeAlB in 9L gliosarcoma
cells. Values are expressed as percent of control uptake (no
inhibitor). Uptake was determined after 30 min incubations
and normalized for dose and number of cells. p-Values
represent comparisons of uptake in the presence of inhibitor
to control uptake for each radiotracer (1-way ANOVA). * ) p
< 0.05, and ** ) p < 0.01. Bars indicate standard error.
a
Reagents: (a) SOCl2, Et3N, toluene or CH2Cl2. (b) NaIO4,
catalytic RuO2, H2O, CH3CN. (c) [18F]HF, K2,2,2, K2CO3, CH3CN,
85 °C and then 6 N HCl, 85 °C.
proceed at 40 °C even with prolonged reaction times (>4
days), the desired intermediate was obtained rapidly
when the solvent was removed under reduced pressure
at 40 °C. The crude amino ester from this procedure was
monoalkylated with bis(4-methoxyphenyl)chloromethane
to provide 12a .
As shown in Scheme 3, the amino alcohol 12b was
also prepared from compound 8. First, 8 was treated
with methyl iodide and sodium hydride in DMF to afford
the N-methyl derivative 10 in quantitative yield. Cata-
lytic hydrogenolysis of 10 provided the alcohol 11, which
was then converted to 12b with p-toluenesulfonic acid
as previously described.
Scheme 4 depicts the formation of the cyclic sulfami-
date precursors 14a ,b and subsequent radiolabeling to
produce the amino acids [18F]5a and [18F]5b. The amino
alcohols 12a ,b were reacted with thionyl chloride in the
presence of triethylamine to form cyclic sulfamidites
13a ,b. Oxidation using sodium periodate with catalytic
ruthenium(IV) oxide provided 14a ,b from 13a ,b, re-
spectively. The precursors 14a ,b are stable for at least
6 months when stored at -10 °C.
unlabeled material in the final product arising from the
precursors is about 1 mg in each case. On the basis of
a 100 mCi yield at the EOS, the minimum ratio of
radiotracer to unlabeled material for both [18F]5a and
[18F]5b is 1 mCi per 10 µg of unlabeled material. This
amount of unlabeled material is comparable to the
amount present in doses of [18F]FDG, which also contain
nonradioactive material arising from the triflate precur-
sor of [18F]FDG.32 In doses of [18F]FDG, the majority of
unlabeled material is comprised of glucose and man-
nose, both of which are not toxic. In the cases of [18F]5a
and [18F]5b, the potential toxicity of the unlabeled
material present in doses must be evaluated prior to
the use of these compounds in human studies.
Am in o Acid Up ta k e Assa ys. To test the hypothesis
that [18F]5a and [18F]5b enter cells predominantly via
the A type amino acid transport system, amino acid
uptake assays using cultured 9L gliosarcoma cells in
the presence and absence of two well-described inhibi-
tors of amino acid transport were performed. N-MeAIB
is a selective competitive inhibitor of the A type amino
acid transport system while 2-amino-bicyclo[2.2.1]-
heptane-2-carboxylic acid (BCH) is commonly used as
an inhibitor for the sodium-independent L type trans-
port system, although this compound also competitively
inhibits amino acid uptake via the sodium-dependent
B0,+ and B0 transport systems.15 The A and L type
amino acid transport systems have been implicated in
the in vivo uptake of radiolabeled amino acids used for
tumor imaging.8,19
Ra d ioch em istr y. Initial attempts to synthesize [18F]-
5a via nucleophilic substitution of the methyl sulfonyl
ester of 9 did not demonstrate measurable 18F incorpo-
ration after prolonged heating. In contrast, the cyclic
sulfamidate precursor 14a provided an average 78%
decay-corrected yield (n ) 4 runs) of [18F]5a in over 99%
radiochemical purity. Likewise, treatment of 14b under
the same conditions gave an average 85% decay-cor-
rected yield (n ) 3 runs) of [18F]5b in over 99%
radiochemical purity. The radiolabeled amino acids were
prepared in a one pot synthesis by treating the precur-
sor 14a or 14b with no-carrier-added [18F]fluoride at 85
°C for 20 min followed by acid hydrolysis at 85 °C for
10 min. The reaction mixture was then passed through
a column containing ion-retardation resin followed by
alumina and C-18 SepPaks. The eluted fractions were
pH 6-7 and suitable for direct use in the rodent studies.
In a representative synthesis, a total of 76 mCi of [18F]-
5b at end of synthesis (EOS) was obtained from 166 mCi
of 18F (end of bombardment, EOB) in a synthesis time
of approximately 90 min.
In the absence of inhibitors, both [18F]5a and [18F]5b
showed similar levels of uptake in 9L gliosarcoma cells,
with intracellular accumulations of 0.43 and 0.50% of
the initial dose per million cells after 30 min of incuba-
tion, respectively. To facilitate the comparison of the
effects of the inhibitors, the data were expressed as
percent uptake relative to the control condition (no
inhibitor) as shown in Figure 1. In the case of [18F]5a ,
BCH blocked 48% of the uptake of activity relative to
controls while N-MeAIB blocked 80% of uptake relative
to controls. The reduction of uptake of [18F]5a by both
BCH and N-MeAIB as compared to controls was sta-
tistically significant (p < 0.05 and p < 0.01, respectively,
While the specific activities of [18F]5a and [18F]5b
were not determined directly, the maximum amount of

Amino Acids for Tumor Imaging with PET
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2243
Ta ble 1. Tissue Distribution of Radioactivity in Normal
Fischer Rats after Injection of [18F]FAMP (5a )a
Ta ble 2. Tissue Distribution of Radioactivity in Normal
Fischer Rats after Injection of [18F]N-MeFAMP (5b)a
tissue
blood
heart
lung
liver
spleen
5 min
30 min
60 min
120 min
tissue
blood
heart
lung
liver
spleen
5 min
30 min
60 min
120 min
0.53 ( 0.03 0.25 ( 0.014 0.22 ( 0.02 0.15 ( 0.005
0.29 ( 0.012 0.29 ( 0.04 0.28 ( 0.013 0.23 ( 0.03
0.55 ( 0.006 0.54 ( 0.12 0.44 ( 0.05 0.37 ( 0.10
0.65 ( 0.05 0.56 ( 0.04 0.50 ( 0.013 0.48 ( 0.03
0.83 ( 0.05 0.54 ( 0.03 0.50 ( 0.04 0.33 ( 0.02
0.61 ( 0.02 0.30 ( 0.003 0.18 ( 0.02 0.10 ( 0.007
0.22 ( 0.01 0.23 ( 0.03 0.20 ( 0.02 0.17 ( 0.02
0.53 ( 0.03 0.50 ( 0.07 0.38 ( 0.07 0.30 ( 0.08
0.79 ( 0.06 0.72 ( 0.09 0.78 ( 0.10 0.58 ( 0.11
0.44 ( 0.06 0.75 ( 0.06 0.68 ( 0.02 0.47 ( 0.04
pancreas 3.46 ( 0.19 2.93 ( 0.20 2.95 ( 0.27 2.48 ( 0.03
kidney
bone
muscle
testis
brain
pancreas 2.73 ( 0.28 3.00 ( 0.23 3.24 ( 0.30 2.88 ( 0.40
kidney
bone
muscle
testis
brain
6.36 ( 0.36 5.59 ( 0.28 4.74 ( 0.15 2.97 ( 0.17
0.27 ( 0.005 0.22 ( 0.02 0.19 ( 0.03 0.13 ( 0.012
0.22 ( 0.013 0.22 ( 0.009 0.26 ( 0.02 0.19 ( 0.003
0.17 ( 0.010 0.10 ( 0.006 0.12 ( 0.006 0.10 ( 0.004
0.04 ( 0.003 0.05 ( 0.004 0.06 ( 0.004 0.05 ( 0.001
8.12 ( 0.62 4.60 ( 0.70 2.73 ( 0.35 1.36 ( 0.27
0.34 ( 0.04 0.32 ( 0.04 0.29 ( 0.04 0.20 ( 0.02
0.16 ( 0.008 0.16 ( 0.003 0.15 ( 0.007 0.13 ( 0.007
0.17 ( 0.004 0.11 ( 0.005 0.08 ( 0.005 0.06 ( 0.005
0.04 ( 0.005 0.04 ( 0.004 0.03 ( 0.003 0.03 ( 0.001
a
a
Values are reported as mean percent dose per gram
(
Values are reported as mean percent dose per gram (
standard error. n ) 4 at each time point.
standard error. n ) 4 at each time point.
by one way analysis of variance (ANOVA)). The mag-
nitude of uptake inhibition of [18F]5a by BCH was less
than that observed with N-MeAIB, but this difference
between inhibitors did not reach statistical significance
in this experiment.
In assays employing [18F]5b, BCH inhibited 33% of
uptake of activity as compared to controls while N-
MeAIB blocked 88% of uptake as compared to controls.
Only the reduction of uptake of [18F]5b by N-MeAIB was
significantly different from control uptake (p < 0.01 by
one way ANOVA), although a trend toward reduction
was observed with BCH. Also, N-MeAIB reduced uptake
of [18F]5b to a greater extent than BCH (p < 0.05 by
one way ANOVA).
2 h study. The brain showed the lowest uptake of
radioactivity, with approximately 0.05% ID/g at all time
points.
The results of the biodistribution study with [18F]5b
in normal rats were very similar to those obtained with
[18F]5a . These results for [18F]5b are depicted in Table
2. The highest uptake was observed in the pancreas and
the kidneys with 2.73 and 8.12% ID/g, respectively, at
5 min. As with [18F]5a , the brain uptake of activity was
very low, with approximately 0.04% ID/g in the brain
at all time points. The low brain uptake of these
compounds is consistent with the observation that the
A type amino transport system is not present at the
intact blood-brain barrier (BBB).33 The lack of signifi-
cant accumulation of radioactivity in bone indicates that
significant in vivo defluorination resulting from me-
tabolism did not occur with either compound during the
2 h studies.
The data obtained from normal rats with both [18F]-
5a and [18F]5b are similar to the reported distributions
of [11C]AIB in rats21 and [14C]AIB in mice,18 suggesting
that these amino acids have similar transport mecha-
nisms in vivo. This observation is consistent with the
A type transport observed for both [18F]5a and [18F]5b
in vitro. In healthy Copenhagen rats, the uptake of [11C]-
AIB at 60 min was highest in the kidneys and pancreas,
with 10.3 and 6.0 mean relative concentrations (mean
RC, calculated from the dose fraction in the tissue
divided by tissue weight multiplied by body weight),
respectively. In contrast, the brain had the lowest mean
RC of [11C]AIB with a value of 0.2. In nude Swiss mice
bearing human melanoma transplants that received
doses of [14C]AIB, similar results were obtained. The
highest mean RC values were observed in the kidneys
and pancreas at 60 min, with values of 3.4 and 8.6,
respectively, while the brain had the lowest mean RC
of organs studied with a value of 0.23. As with [18F]5a
and [18F]5b, the uptake of radioactivity in the pancreas
and kidneys was rapid in these studies of AIB biodis-
tribution, with mean RC values in both tissues greater
than 1.8 within 5-15 min postinjection. In both studies
of AIB, moderate liver uptake of activity was observed,
while the mean RC in blood and muscle was low at 60
min.
Taken together, these inhibition studies indicate that
[18F]5a and [18F]5b are substrates for the A type amino
acid transport system in 9L gliosarcoma cells based on
the inhibition of uptake of both compounds in the
presence of N-MeAIB. Additionally, [18F]5a is also a
substrate in vitro for at least one non-A transport
system, possibly system L, based on the inhibition of
uptake by BCH. The data indicate that [18F]5b is a more
selective substrate for the A type amino transport
system than [18F]5a , consistent with the increased
selectivity of N-MeAIB for A type transport relative to
AIB.22,23 Because [18F]5a and [18F]5b were evaluated as
racemic mixtures, it is possible that the enantiomers of
these compounds differ in their specificity for the
various amino acid transport systems. A more detailed
analysis of the biological transport properties of these
radiotracers and their single enantiomers in a panel of
other tumor cell lines is in progress.
Biod istr ibu tion in Nor m a l Ra ts. The results of the
biodistribution studies with [18F]5a in normal rats are
presented in Table 1. At 5 min after tail vein injection
of [18F]5a , both the pancreas and the kidneys showed
significantly higher uptake of radioactivity than the
other tissues studied (p < 0.001 by one way ANOVA),
with 3.46 and 6.36% of the injected dose per gram of
tissue (% ID/g), respectively. The activity in these
tissues remained above the activity in other tissues (p
< 0.001 by one way ANOVA at all time points), with
2.48% ID/g in the pancreas and 2.97% ID/g in the
kidneys at 120 min. The liver showed moderate uptake
of activity, with 0.65% ID/g at 5 min, which decreased
to 0.48% ID/g after 120 min. Other tissues studied,
including heart, lung, bone, blood, muscle, and testis,
showed relatively low uptake of radioactivity at 5 min
(<0.55% ID/g), which decreased over the course of the
High pancreatic uptake of radioactivity has been
reported for a number of other 11C-labeled amino acids
in rats, including Met, L-[11C]leucine, and 1-amino-
cyclopentane-1-[11C]carboxylic acid.34 The pancreatic
uptake ranged from approximately 3 to 5% ID/g at 60

2244 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
McConathy et al.
Ta ble 3. Tissue Distribution of Radioactivity in
Ta ble 4. Tissue Distribution of Radioactivity in
Tumor-Bearing Fischer Rats after Injection of [18F]FAMP (5a )a
Tumor-Bearing Fischer Rats after Injection of [18F]N-MeFAMP
(5b)a
tissue
blood
heart
lung
5 min
60 min
120 min
tissue
blood
heart
lung
5 min
60 min
120 min
0.72 ( 0.05
0.40 ( 0.04
0.84 ( 0.03
0.77 ( 0.22
0.72 ( 0.06
5.31 ( 0.68
8.66 ( 1.27
0.38 ( 0.04
0.39 ( 0.02
0.22 ( 0.02
0.26 ( 0.03
0.33 ( 0.06
0.38 ( 0.04
0.66 ( 0.16
0.55 ( 0.08
2.86 ( 0.34
6.20 ( 1.45
0.25 ( 0.05
0.31 ( 0.03
0.14 ( 0.02
0.16 ( 0.01
0.22 ( 0.02
0.24 ( 0.03
0.25 ( 0.02
0.32 ( 0.02
1.42 ( 0.17
3.64 ( 0.26
0.16 ( 0.01
0.21 ( 0.01
0.11 ( 0.003
0.69 ( 0.04
0.34 ( 0.02
0.61 ( 0.04
0.94 ( 0.12
0.47 ( 0.04
2.95 ( 0.58
9.33 ( 0.36
0.33 ( 0.04
0.22 ( 0.011
0.19 ( 0.008
0.17 ( 0.02
0.25 ( 0.03
0.26 ( 0.03
0.85 ( 0.17
0.45 ( 0.06
2.42 ( 0.27
2.79 ( 0.31
0.23 ( 0.04
0.19 ( 0.02
0.08 ( 0.008
0.10 ( 0.007
0.19 ( 0.02
0.19 ( 0.011
0.41 ( 0.06
0.40 ( 0.02
1.74 ( 0.24
1.59 ( 0.19
0.19 ( 0.02
0.16 ( 0.012
0.08 ( 0.005
liver
spleen
pancreas
kidney
bone
muscle
testis
brain
tumor
tumor:brain 26:1
ratio
liver
spleen
pancreas
kidney
bone
muscle
testis
brain
tumor
tumor:brain 40:1
ratio
0.035 ( 0.002* 0.054 ( 0.01** 0.050 ( 0.004†
0.91 ( 0.05*
1.96 ( 0.10**
36:1
1.87 ( 0.2†
37:1
0.032 ( 0.002* 0.022 ( 0.006** 0.020 ( 0.005†
1.29 ( 0.28*
2.28 ( 0.16**
104:1
1.94 ( 0.12†
97:1
a
Values are reported as mean percent dose per gram
(
a
standard error. n ) 4 at 5 min, n ) 5 at 60 min, and n ) 4 at 120
min; p values were determined using a two-tailed t-test for
pairwise comparisons. * ) p < 0.001, ** ) p < 0.001, and † ) p <
0.003.
Values are reported as mean percent dose per gram (
standard error. n ) 4 at each time point; p values were determined
using a two-tailed t-test for pairwise comparisons. * ) p < 0.02,
** ) p < 0.001, and † ) p < 0.001.
brain and 1.05% ID/g in the tumor tissue, demonstrat-
ing the high levels of [18F]FDG uptake in normal brain
tissue. At 60 min postinjection, [18F]FACBC showed a
7:1 tumor to brain ratio with 1.72% ID/g in tumor tissue
vs 0.26% ID/g in normal brain. A similar ratio of tumor
to brain uptake of radiotracer was seen with [18F]-
FACBC in a PET scan of a human volunteer with
biopsy-confirmed glioblastoma multiforme (6:1 ratio at
20 min postinjection), suggesting that this rodent model
is useful in predicting imaging properties of radiolabeled
amino acids in human patients with brain tumors.
As in the normal rats receiving [18F]5a or [18F]5b,
high levels of uptake occurred in the pancreas and
kidneys of the tumor-bearing rats. Additionally, both
compounds had high uptake in tumor tissue but rela-
tively low uptake in other tissues examined including
heart, lung, muscle, liver, bone, and testis. Interestingly,
[18F]FACBC showed lower uptake in the kidneys in the
same animal model (0.60% ID/g at 60 min), which may
reflect less reuptake from the glomerular filtrate but
higher uptake in the liver (1.70% ID/g at 60 min).14 On
the basis of the rodent data, the pancreas, kidneys, and
bladder would be predicted to bear the highest dosim-
etry burden in human studies employing [18F]5a or [18F]-
5b. The low uptake in other normal tissues suggests
that both [18F]5a and [18F]5b might be suitable for
imaging tumors exhibiting high uptake of these amino
acids in locations other than the brain. For example,
the tumor to muscle ratios obtained at 60 min were 6.3:1
for [18F]5a and 12:1 for [18F]5b, while ratios of 5.3:1 for
[18F]FDG and 4.2:1 for [18F]FACBC were observed at
this time point.14 Because both [18F]5a and [18F]5b were
evaluated as racemic mixtures, it is possible that the
single enantiomers of [18F]5a and [18F]5b would exhibit
different biodistribution profiles. If one enantiomer has
superior in vivo properties for tumor imaging, using it
would be advantageous in terms of both radiation
dosimetry and interpretation of tissue uptake of radio-
activity. The isolation and evaluation of the R and S
enantiomers of both [18F]5a and [18F]5b are underway.
A comparison of the uptake of radioactivity in tumor
tissue and brain tissue after [18F]5a and [18F]5b ad-
ministration is depicted in Figure 2a,b. The higher
ratios of tumor to brain uptake obtained with [18F]5b
vs [18F]5a appear to be due to lower brain uptake of
min in this study of these compounds. Similarly, [18F]-
FACBC showed 3.4% ID/g at 60 min in normal Fischer
rats. The similarity between the biodistribution patterns
of [18F]5a , [18F]5b, and radiolabeled AIB, and in par-
ticular the high pancreatic and low brain uptake of
radioactivity, prompted us to evaluate these compounds
in tumor-bearing rats.
Biod istr ibu tion in Tu m or -Bea r in g Ra ts. The tis-
sue distribution of radioactivity after tail vein injection
of [18F]5a in the normal tissues of tumor-bearing rats
was similar to that seen in normal rats and is presented
in Table 3. Tumor uptake of radioactivity at 5, 60, and
120 min after injection was 0.91, 1.96, and 1.87% ID/g,
respectively, while uptake in normal brain tissue con-
tralateral to the tumor was approximately 0.05% ID/g
at each time point. The higher uptake of activity by the
tumor vs normal brain was statistically significant at
each time point (p < 0.001 at 5 and 60 min and p <
0.003 at 120 min by two-tailed paired t-tests). The
resulting ratios of tumor uptake to normal brain uptake
were 26:1, 36:1, and 37:1 at 5, 60, and 120 min,
respectively.
The results from the same study conducted with [18F]-
5b in tumor-bearing rats are summarized in Table 4
and demonstrated tumor uptake of 1.29, 2.28, and 1.94%
ID/g at 5, 60, and 120 min postinjection, respectively.
At each time point, the higher uptake of activity in the
tumor vs normal brain tissue was statistically signifi-
cant (p < 0.02 at 5 min and p < 0.001 at 60 and 120
min by two-tailed paired t-tests). The ratios of tumor
uptake to normal brain uptake at 5, 60, and 120 min
obtained with [18F]5b were 40:1, 104:1, and 97:1,
respectively. Because of the relatively long intervals
between time points, it is possible that the highest
tumor to brain ratio was not observed for [18F]5a or [18F]-
5b. Imaging studies in nonhuman primates and in
human cancer patients will provide more detailed
information regarding the biodistribution and kinetics
of tracer uptake in normal and neoplastic tissue.
For both [18F]5a and [18F]5b, the ratios of tumor to
brain uptake of activity are higher than those reported
for [18F]FDG and [18F]FACBC in the same rodent tumor
model.14 In the case of [18F]FDG, the tumor to brain
ratio was 0.8:1 at 60 min with 1.30% ID/g in normal

Amino Acids for Tumor Imaging with PET
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2245
F igu r e 2. (a) Comparison of activity in tumor tissue after injection of [18F]FAMP (5a ) and [18F]N-MeFAMP (5b). Tissues were
compared at each time point by two-tailed t-test. No significant differences were detected. Bars indicate standard error. (b)
Comparison of activity in normal brain after injection of [18F]FAMP (5a ) and [18F]N-MeFAMP (5b). Tissues were compared at
each time point by two-tailed t-test. * ) p < 0.03, and ** ) p < 0.003. Bars indicate standard error.
activity with [18F]5b rather than higher tumor uptake
of activity. No statistically significant differences were
detected between the two compounds when comparing
the uptake of activity in the tumor at the three time
points studied (see Figure 2a). This observation is
consistent with the amino acid uptake assay in which
cultured 9L gliosarcoma cells accumulated [18F]5a and
[18F]5b in similar amounts. However, at both 60 and
120 min postinjection, the uptake of radioactivity in the
normal brain tissue of rats receiving [18F]5b was
significantly less than in animals receiving [18F]5a . At
60 min after [18F]5a injection, the brain uptake was
0.054 vs 0.022% ID/g in animals receiving [18F]5b (p <
0.03 by a two-tailed t-test); at 120 min after [18F]5a
injection, the brain uptake was 0.050 vs 0.020% ID/g in
animals receiving [18F]5b (p < 0.003 by a two-tailed
t-test). The magnitude of the difference in brain uptake
between the compounds is enough to account for the
difference in tumor to brain uptake ratios at these time
points.
It is likely that the difference in normal brain uptake
of activity is due to the higher selectivity of [18F]5b vs
[18F]5a for the A type amino acid transport system,
which is not active at the normal BBB.33 Some of the
other amino acid transport systems, such as the L type
transport system, are active at the normal BBB19 and
could potentially mediate uptake of these radiolabeled
amino acids. The A type amino acid transport system
tolerates amino acid substrates with an N-methyl group
while the other amino acid transport systems generally
do not,15,22,23 and the uptake inhibition assays discussed
earlier suggest that [18F]5b is a more selective substrate
for A type transport than [18F]5a . The lower uptake of
activity observed with [18F]5b relative to [18F]5a in
normal brain but not in tumor or pancreatic tissue is
consistent with the increased selectivity of N-methyl
amino acids for the A type amino acid transport system.
If [18F]5a and [18F]5b were entering normal brain by
diffusion alone, the more lipophilic [18F]5b would be
expected to show higher brain uptake than [18F]5a .
The high uptake of [18F]5a and [18F]5b in tumor tissue
combined with low uptake in normal brain accounts for
the high tumor to brain ratios observed with these
compounds, but the low uptake across the normal BBB
may also present difficulties in PET imaging studies of
brain tumors. Disruptions of the BBB due to nonneo-
plastic processes may lead to increased uptake of
radioactivity in lesions relative to normal brain tissue.
Conversely, low grade neoplasms may have normal
BBBs that do not permit these radiotracers to reach the
tumor cells. These potential problems must be ad-
dressed as the compounds undergo further evaluation.
However, subtle alterations in BBB metabolism and
transport may precede gross disruption of the BBB in
low grade tumors, and tumors outside the central
nervous system would not be liable to this effect.
Con clu sion s. The work presented here demonstrates
that both [18F]5a and [18F]5b can be produced in high
radiochemical yield (>78% EOB) and high radiochemi-
cal purity from stable precursors and are potentially
valuable agents for imaging brain tumors with PET. The
synthetic strategy developed for [18F]5a provides an
efficient route for preparing radiolabeled primary amines
with 18F in the â position. Uptake inhibition studies
using 9L gliosarcoma cells demonstrated that both
compounds are substrates for the A type amino acid
transport system, and these compounds represent the
first report of 18F-labeled amino acids that undergo
significant uptake via A type transport. Biodistribution
studies with both compounds showed rapid and persis-
tent accumulation of radioactivity in rodent brain
tumors with an excellent signal to background ratio.
Injection of [18F]5b led to ratios of 104:1 and 97:1 in
tumor vs normal brain at 60 and 120 min, respectively,
while injection of [18F]5a led to ratios of 36:1 and 37:1
at the same time points. With the exception of the
pancreas and kidneys, other tissues studied including
muscle, lung, heart, and liver showed relatively low
uptake of radioactivity. Studies are currently underway
to determine the toxicity, metabolic stability, and radia-
tion dosimetry associated with [18F]5a and [18F]5b
administration and to determine the transport proper-
ties of the isolated R and S enantiomers of these
compounds.
Exp er im en ta l Section
Ch em istr y. All reagents used were obtained from com-
mercially available sources. Solvents used in reactions were
purchased from Aldrich Chemicals while solvents for chroma-
tography were obtained from VWR. Melting points are uncor-
rected and were determined in capillary tubes on an Electro-
chemical 9100 apparatus. 1H nuclear magnetic resonance
(NMR) spectra were recorded on a Varian spectrometer at 400
MHz unless otherwise indicated and referenced to the NMR
solvent (chemical shifts in δ values, J in Hz). Mass spectra

2246 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
McConathy et al.
were determined on a VG 70-S double-focusing mass spec-
trometer using high-resolution electron ionization. Elemental
analyses were performed by Atlantic Microlabs, Inc. and were
within ( 0.4% unless otherwise stated. The phrase “usual work
up” refers to the use of anhydrous magnesium sulfate followed
by concentration under reduced pressure. The compounds
3-benzyloxypropanone35 and bis(4-methoxyphenyl)chloro-
methane36 were prepared according to literature procedures.
The target compounds 5a ,b were prepared as racemic mixtures
in both their fluorine-18 and fluorine-19 forms.
2-Am in o-2-cya n o-3-flu or op r op a n e (1). To a solution of
1 equiv of NH4Cl (700 mg) and 1 equiv of KCN (853 mg) in 10
mL of H2O was added fluoroacetone (1.0 g, 13.1 mmol) in 3
mL of H2O. After it was stirred overnight at room temperature,
the reaction mixture was basified with 10 mL of 1 N NaOH
and extracted with 5 × 20 mL of Et2O. Usual work up afforded
the aminonitrile 1 as a clear oil (510 mg, 38%), which was used
without further purification. 1H NMR (CDCl3), 300 MHz: δ
1.48 (3H, d, J ) 2.1), 4.17-4.33 (1H, m), 4.33-4.49 (1H, m).
HCl and heated to 70 °C for 3 h. The resulting solution was
evaporated under reduced pressure, and the solid was washed
with 2 × 10 mL of Et2O to provide 5b (16 mg, 91%) as a white
1
solid; decomp 178-181 °C. H NMR (D2O): δ 1.47-1.48 (3H,
m), 2.73 (3H, s), 4.64-4.90 (2H, m). Anal. (C5H11ClFNO2) C,
H, N.
1-Meth yl-1-(ben zyloxym eth yl)h yd a n toin (6). To a solu-
tion of 3-benzyloxypropanone (5.7 g, 34.7 mmol) in 180 mL of
1:1 EtOH:H2O was added 10 equiv of ammonium carbonate
(33 g) followed by 4 equiv of ammonium chloride (7.42 g). After
the mixture was stirred at room temperature for 30 min, a
4.5 equiv portion of potassium cyanide (10.2 g) was added, and
the reaction mix was stirred for 48 h at room temperature.
The solvent was evaporated under reduced pressure, and the
resulting solid was washed with 3 × 30 mL of water to afford
the hydantoin 6 (6.4 g, 79% yield) as a yellowish solid suitable
for use in the next step. Analytically pure samples were
obtained via chromatography on silica gel (EtOAc); mp 94.5-
1
96 °C (EtOAc). H NMR (CDCl3): δ 1.43 (3H, s), 3.47 (1H, d,
2-[N-(ter t-Bu t oxyca r b on yl)a m in o]-3-flu or o-2-m et h yl-
p r op a n oic Acid (2). The crude aminonitrile 1 (510 mg, 5.00
mmol) was refluxed overnight in 20 mL of 6 N HCl, and the
solvent was removed under reduced pressure. The crude white
solid was dissolved in 20 mL of 85:15 CH3OH:Et3N and treated
with 2.6 equiv of di-tert-butyl dicarbonate (2.8 g). After the
mixture was stirred overnight, the solvent was removed under
reduced pressure, and the resulting paste was stirred in ice-
cold 1:1 EtOAc:0.2 N aqueous HCl for 5 min. The aqueous layer
was further extracted with 2 × 50 mL of ice-cold EtOAc. The
combined organic layers were washed with 2 × 50 mL of H2O
followed by usual work up. Purification by silica gel column
chromatography (10% CH3OH in CH2Cl2) provided 2 (767 mg,
69%) as a light yellow solid suitable for use in the next step.
Analytically pure samples were obtained using the same
procedure followed by further purification by silica gel column
chromatography (1:3 EtOAc:hexane followed by 1:1 EtOAc:
hexane) to provide the N-Boc acid 2 as a white solid; mp 122-
123 °C (EtOAc/hexane). 1H NMR (CDCl3): δ 1.45 (9H, s), 1.56
(3H, d, J ) 1.6), 4.74 (2H, d, J ) 46.8), 5.30 (1H, broad s).
Anal. (C9H16FNO4) C,H,N.
J ) 9.6), 3.62 (1H, d, J ) 9.6), 4.50-4.60 (2H, m), 5.37 (1H,
broad s), 7.27-7.38 (5H, m) 7.45 (1H, broad s). Anal.
(C12H14N2O3) C, H, N.
3-B e n zy lo x y -2-[N -(t er t -b u t o x y c a r b o n y l)a m in o ]-2-
m eth ylp r op a n oic Acid (7). A suspension of the hydantoin
6 (2.0 g, 8.5 mmol) in 55 mL of 5 M NaOH was heated at 180
°C overnight in a sealed steel vessel. After it was cooled, the
reaction mix was brought to pH 7 using concentrated HCl, and
the solvent was evaporated under reduced pressure. The white
solid was extracted with 4 × 20 mL of hot EtOH, and the
combined extracts were concentrated under reduced pressure.
The resulting residue was dissolved in 50 mL of 9:1 CH3OH:
Et3N and treated with 2 equiv of di-tert-butyl dicarbonate (3.72
g) overnight at room temperature. The reaction mixture was
concentrated under reduced pressure and purified by silica gel
column chromatography (5% CH3OH in CH2Cl2) to afford 7
as an off-white solid (1.76 g, 67%); mp 112.5-114.5 °C (CH3-
OH/CH2Cl2). 1H NMR (CDCl3): δ 1.45 (9H, s), 1.46 (3H, s),
3.72 (1H, d, J ) 9.2), 3.81 (1H, d, J ) 9.6), 4.59 (2H, d, J )
1.6), 5.44 (1H, broad s), 7.30-7.38 (5H, m). Anal. (C16H23NO5)
C, H, N.
2-[N-(ter t-Bu t oxyca r b on yl)a m in o]-3-flu or o-2-m et h yl-
p r op a n oic Acid ter t-Bu tyl Ester (3). The N-Boc acid 2 (767
mg, 3.46 mmol) in 10 mL of dry CH2Cl2 was stirred overnight
with 3 equiv of tert-butyl-2,2,2-tricholoracetimdate (2.27 g).
After it was concentrated under reduced pressure, the crude
product was purified by silica gel column chromatography (5%
EtOAc in hexane) to provide 3 (510 mg, 53%) as a white solid;
3-B e n zy lo x y -2-[N -(t er t -b u t o x y c a r b o n y l)a m in o ]-2-
m eth ylp r op a n oic Acid ter t-Bu tyl Ester (8). To a solution
of the N-Boc carboxylic acid 7 (1.6 g, 5.17 mmol) in 15 mL of
CH2Cl2 at room temperature was added a 3 equiv portion of
tert-butyl-2,2,2-trichloroacetimidate (3.4 g). After it was stirred
overnight at room temperature, the solvent was evaporated
under reduced pressure. Purification via silica gel column
chromatography (15% EtOAc in hexane) afforded 8 as a
colorless oil (1.71 g, 90%). 1H NMR (CDCl3): δ 1.44 (9H, s),
1.45 (9H, s), 1.48 (3H, s), 3.66 (1H, d, J ) 8.8), 3.79-3.82 (1H,
broad d), 4.48-4.58 (2H, m), 5.51 (1H, broad s), 7.28-7.35 (5H,
m). Anal. (C20H31NO5) C, H, N.
1
mp 41-42 °C (EtOAc/hexane). H NMR (CDCl3): δ 1.44 (9H,
s), 1.47 (3H, d, J ) 2.4), 1.48 (9H, s), 4.57-4.85 (2H, m), 5.34
(1H, broad s). Anal. (C13H24FNO4) C, H, N.
2-[N-(ter t-Bu t oxyca r b on yl)m et h yla m in o]-3-flu or o-2-
m eth ylp r op a n oic Acid ter t-Bu tyl Ester (4). To a solution
of 3 (200 mg, 0.72 mmol) in dry DMF under an argon
atmosphere was added 8 equiv of CH3I (0.36 mL) followed by
2 equiv of 95% NaH (37 mg). The reaction mixture was stirred
overnight at room temperature. The reaction mix was added
to 15 mL of H2O and extracted with 3 × 15 mL of Et2O. The
combined organic layers were washed with 3 × 20 mL H2O
followed by the usual work up. Purification of the crude
product via silica gel column chromatography (7.5% EtOAc in
hexane) afforded the methylated species 4 (181 mg, 86%) as a
colorless oil. 1H NMR (CDCl3): δ 1.44 (9H, s), 1.46 (9H, s),
2-[N-(ter t-Bu toxyca r bon yl)a m in o]-3-h yd r oxy-2-m eth -
ylp r op a n oic Acid ter t-Bu tyl Ester (9). A suspension of the
benzyl ether 8 (540 mg,1.48 mmol) and 10% Pd-C (130 mg)
in 20 mL of CH3OH was stirred under an H2 atmosphere
overnight. The reaction mixture was filtered over Celite, and
the filtrate was concentrated under reduced pressure. Purifi-
cation via silica gel column chromatography (30% EtOAc in
hexane) provided a quantitative yield of 9 (407 mg, 100%) as
a colorless solid; mp 44-45 °C (EtOAc/hexane). 1H NMR
(CDCl3): δ 1.437 (3H, s), 1.442 (9H, s), 1.48 (9H, s), 3.72 (1H,
d, J ) 11.2), 4.00 (1H, d, J ) 11.2), 5.32 (1H, broad s). Anal.
(C13H25NO5) C, H, N.
1.51 (3H, s), 2.94 (3H, s), 4.51-5.04 (2H, m). Anal. (C14H26
FNO4) C, H, N.
-
F AMP (5a ), Hyd r och lor id e Sa lt. The N-Boc amino acid
2 (30 mg, 0.14 mmol) was suspended in 0.3 mL of 4 N HCl
and heated to 50 °C for 90 min. The resulting homogeneous
solution was evaporated under reduced pressure to provide
the crude HCl salt of the amino acid. The solid was washed
with 2 × 10 mL of Et2O to provide 5a (18 mg, 84%) as a white
solid; decomp 204-206 °C. 1H NMR (D2O): δ 1.52 (3H, s),
4.55-4.91 (2H, m). Anal. (C4H9ClFNO2) C, H, N.
3-Ben zyloxy-2-[N-(ter t-bu toxyca r bon yl)m eth yla m in o]-
2-m eth ylp r op a n oic Acid ter t-Bu tyl Ester (10). The same
procedure used to obtain 4 was employed using 745 mg of 8
(2.04 mmol). The crude product was purified via silica gel
column chromatography (10% EtOAc in hexane) to provide 10
(770 mg, 99%) as a colorless oil. 1H NMR (CDCl3): δ 1.43 (18H,
s), 1.50 (3H, s), 2.96 (3H, s), 3.67 (1H, d, J ) 10), 4.04 (1H,
broad s), 4.52 (2H, s), 7.24-7.34 (5H, m). Anal. (C21H33NO5)
C, H, N.
N-MeF AMP (5b), Hyd r och lor id e Sa lt. The N-Boc tert-
butyl ester 4 (30 mg, 0.10 mmol) was stirred in 0.6 mL of 6 N

Amino Acids for Tumor Imaging with PET
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2247
2-[N-(ter t-Bu toxyca r bon yl)m eth yla m in o]-3-h yd r oxy-2-
m eth ylp r op a n oic Acid ter t-Bu tyl Ester (11). The same
hydrogenolysis conditions used to convert 8 to 9 were applied
to a 350 mg portion of 10 (0.92 mmol) to afford 11 (266 mg,
mL of EtOAc. The organic layers were combined and washed
with 20 mL of brine followed by usual work up. Purification
by silica gel column chromatography (15% EtOAC in hexane)
provided a 1.8:1 mixture of diastereomers 13b (76 mg, 58%)
as a colorless oil. The mixture of diastereomers was used
immediately in the next step as the compounds decomposed
over time. The diastereomers could be separated in small
amounts using the same chromatography conditions. 1H NMR
(CDCl3) for major diastereomer: δ 1.47 (9H, s), 1.56 (3H, s),
2.86 (3H, s), 4.55 (1H, d, J ) 9.2), 4.73 (1H, d, J ) 8.8). Anal.
(C9H17NO4S) Calcd: C, 45.94; H, 7.28; N, 5.95. Found: C,
1
100%) as a colorless oil. H NMR (CDCl3): δ 1.45-1.46 (21H,
m), 2.88 (3H, s), 3.50 (1H, d, J ) 14.8 Hz), 4.02 (1H, J ) 15.6
Hz). Anal. (C14H27NO5) C, H, N.
3-Hyd r oxy-2-(N-[bis(4-m eth oxyp h en yl)m eth yl]a m in o)-
2-m eth ylp r op a n oic Acid ter t-Bu tyl Ester (12a ). To a
solution of the alcohol 9 (100 mg, 0.36 mmol) in 2 mL of diethyl
ether was added 1 equiv of p-toluenesulfonic acid monohydrate
(69 mg) dissolved in 6 mL of EtOH. The reaction mixture was
concentrated under reduced pressure at 40 °C, and the residue
was dissolved in 6 mL of EtOH and concentrated again. This
process was repeated four times, at which time no starting
material was present on thin-layer chromatography (TLC)
analysis. The resulting white solid was suspended in 3 mL of
CH2Cl2 and treated with 4.5 equiv of triethylamine (0.23 mL)
followed by 1 equiv of bis(4-methoxyphenyl)chloromethane (95
mg). The reaction mixture was stirred for 1 h at room
temperature. The solution was then partitioned between 10
mL of EtOAc and 10 mL of H2O. The aqueous layer was
extracted with 10 mL of EtOAc followed by the usual work up
of the combined organic layers. Purification by silica gel
column chromatography (20% EtOAc in hexane) provided the
amino ester 12a as a colorless oil (107 mg, 73% from 9). 1H
NMR (CDCl3): δ 1.17 (3H, s), 1.46 (9H, s), 3.35 (1H, d, J )
11.2), 3.44 (1H, d, J ) 11.2), 3.76 (3H, s), 3.77 (3H, s), 4.82
1
45.10; H, 7.59; N, 5.64. H NMR (CDCl3) for minor diastere-
omer: δ 1.44 (3H, s), 1.49 (9H, s), 2.91 (3H, s), 4.03 (1H, d, J
) 8.0), 5.22 (1H, d, J ) 8.4). Anal. (C9H17NO4S) Calcd: C,
45.94; H, 7.28; N, 5.95. Found: C, 46.48; H, 7.41; N, 5.78.
3-[Bis(4-m eth oxyph en yl)m eth yl]-4-m eth yl-1,2,3-oxath i-
a zolid in e-4-ca r boxylic Acid ter t-Bu tyl Ester 2,2-Dioxid e
(14a ). A solution of the diastereomeric sulfamidites 13a (97
mg, 0.22 mmol) in 4 mL of CH3CN was cooled in an ice bath
and treated successively with 1.1 equiv of NaIO4 (51 mg), a
catalytic amount of RuO2‚H2O (∼1 mg), and 2.4 mL of H2O.
After 5 min of stirring, the ice bath was removed, and the
reaction was continued for 20 min. The reaction mixture was
diluted in 10 mL of EtOAc and washed with 10 mL of
saturated NaHCO3 solution. The aqueous layer was extracted
with 2 × 10 mL of EtOAc, and the combined organic layers
were washed with 10 mL of brine followed by usual work up.
The crude product was purified by silica gel column chroma-
tography (30% EtOAc in hexane) to provide the cyclic sul-
famidate 14a as a light yellow solid (90 mg, 89%); mp 143.5-
145 °C (EtOAc/hexane). 1H NMR (CDCl3): δ 1.29 (3H, s), 1.51
(9H, s), 3.77 (3H, s), 3.80 (3H, s), 4.16 (1H, d, J ) 8.8), 4.73
(1H, d, J ) 8.8), 5.98 (1H, s), 6.82-6.89 (4H, m), 7.38-7.44
(4H). Anal. (C23H29NO7S) C, H, N.
(1H, s), 6.80-6.84 (4H, m), 7.27-7.31 (4H, m). Anal. (C23H31
NO5) C, H, N.
-
3-Hyd r oxy-2-m eth yl-2-(m eth yla m in o)p r op a n oic Acid
ter t-Bu tyl Ester (12b). A 255 mg portion of alcohol 11 (0.88
mmol) was treated with 1 equiv p-toluenesulfonic acid (167
mg) as described in the preparation of 12a . The resulting solid
was added to 15 mL of 10% Na2CO3 and extracted with 3 ×
15 mL of EtOAc. The combined organic layers were subject to
the usual work up. Purification via silica gel column chroma-
tography (10% CH3OH in CH2Cl2) afforded 12b (115 mg, 69%)
3,4-Dim eth yl-1,2,3-oxa th ia zolid in e-4-ca r boxylic Acid
ter t-Bu tyl Ester 2,2-Dioxid e (14b). The same reaction
conditions used to obtain 14a were applied to 42 mg of 13b
(0.18 mmol), providing 14b (42 mg, 94%) as a white solid; mp
1
1
as a colorless oil. H NMR (CDCl3): δ 1.24 (3H, s), 1.48 (9H,
54-55 °C (EtOAc/hexane). H NMR (CDCl3): δ 1.50 (9H, s),
s), 2.32 (3H, s), 3.52 (1H, d, J ) 10.8), 3.64 (1H, d, J ) 10.8).
HRMS calcd for C9H19NO3, 189.13649; found, 189.13627. Anal.
(C9H19NO3) Calcd: C, 57.12; H, 10.12; N, 7.40. Found: C,
55.87; H, 10.05; N, 7.18.
1.52 (3H, s), 2.93 (3H, s), 4.22 (1H, d, J ) 8.8), 4.88 (1H, d, J
) 8.8). Anal. (C9H17NO5S) C, H, N.
P r ep a r a tion of 5a (F AMP ) Via 14a . To a solution of the
cyclic sulfamidate 14a (130 mg, 0.28 mmol) in CH3CN (4 mL)
was added 3 equiv of tetrabutylammonium fluoride (1.0 M in
tetrahydrofuran (THF)), and the resulting solution was stirred
overnight at room temperature. The reaction mix was concen-
trated under reduced pressure, and the residue was treated
with 5 mL of 3 N HCl at 85 °C for 1 h. After the mixture was
cooled, the aqueous solution was washed with 5 mL of ether
and then brought to pH 7 with 6 N NaOH. The solvent was
removed under reduced pressure, and the resulting white solid
was dissolved in 9:1 CH3OH:Et3N. To this solution was added
a 2 equiv portion of (Boc)2O (122 mg), and the reaction mixture
was stirred overnight at room temperature. The work up and
purification were performed as previously described to provide
the product 5a (25 mg, 40%), which had the same 1H NMR
spectrum as the product obtained via the aminonitrile route.
3-[Bis(4-m eth oxyph en yl)m eth yl]-4-m eth yl-1,2,3-oxath i-
a zolid in e-4-ca r boxylic Acid ter t-Bu tyl Ester 2-Oxid e
(13a ). A solution of the amino alcohol 12a (105 mg, 0.26 mmol)
and 2.2 equiv of triethylamine (80 µL) in 8 mL of toluene under
an argon atmosphere was cooled in an ice bath followed by
the dropwise addition of 1.1 equiv of thionyl chloride (34 mg)
in 1 mL of toluene. After 15 min, the ice bath was removed,
and the reaction was continued for 10 min. The reaction mix
was partitioned between 10 mL of EtOAc and 10 mL of H2O.
The aqueous layer was further extracted with 3 × 10 mL of
EtOAc. The organic layers were combined and washed with
20 mL of brine followed by usual work up. Silica gel column
chromatography (25% EtOAc in hexane) afforded a 1.6:1
mixture of cyclic sulfamidite diastereomers 13a as a colorless
1
Ra d iosyn th esis of [18F ]5a (F AMP ) a n d
[
18F ]5b (N-
oil (97 mg, 83%). H NMR (CDCl3) for major diastereomer: δ
MeF AMP ). The same conditions were used to prepare [18F]-
5a from 14a and [18F]5b from 14b. To a Wheaton vial
containing 150-200 mCi of no-carrier-added [18F]HF (20 µA,
10-15 min bombardment, theoretical specific activity of 1.7
Ci/nmole) in 350 µL of [18O]H2O were added a 1 mL solution
of 10 mg K222 Kryptofix and 1 mg of K2CO3 in CH3CN. The
solvent was removed at 115 °C with argon gas flow, and an
additional 1 mL of CH3CN was added followed by evaporation
with argon flow. This drying was repeated a total of three
times to remove residual H2O. A 1-2 mg portion of the cyclic
sulfamidate precursor 14a ,b in 1 mL of dry CH3CN was added
to the vial, and the reaction mix was heated at 85 °C for 20
min. The solvent was removed at 115 °C with argon gas flow,
and the intermediate product was treated with 0.5 mL of 6 N
HCl at 85 °C for 10 min. The solution of radiolabeled amino
acid was diluted in 1-2 mL of H2O and eluted in H2O through
1.32 (9H, s), 1.37 (3H, s), 3.78 (3H, s), 3.79 (3H, s), 4.23 (1H,
d, J ) 8.4), 5.34 (1H, d, J ) 8.8), 5.91 (1H, s), 6.83-6.86 (4H,
1
m), 7.17-7.20 (2H, m), 7.38-7.41 (2H, m). H NMR (CDCl3)
for minor diastereomer: δ 1.21 (3H, s), 1.53 (9H, s), 3.77 (3H,
s), 3.81 (3H, s), 4.67 (2H, s), 5.74 (1H, s), 6.82-6.91 (4H, m),
7.33-7.36 (2H, m), 7.51-7.54 (2H, m). Anal. for mixture of
diastereomers: (C23H29NO6S) C, H, N.
3,4-Dim eth yl-1,2,3-oxa th ia zolid in e-4-ca r boxylic Acid
ter t-Bu tyl Ester 2-Oxid e (13b). A solution containing a 103
mg portion of 12b (0.55 mmol) and a 2.2 equiv portion of
triethylamine (0.17 mL) in 2 mL of CH2Cl2 was added dropwise
to a solution of 1.1 equiv thionyl chloride (72 mg) in 2 mL of
dry CH2Cl2 under argon at -78 °C. The reaction mix was
allowed to warm to room temperature overnight. The reaction
mix was partitioned between 10 mL of EtOAc and 10 mL of
H2O. The aqueous layer was further extracted with 2 × 10

2248 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
McConathy et al.
a 7 mm × 120 mm column of ion-retardation resin (Bio Rad
AG11A8 50-100 mesh) in series with 2 Alumina N SepPaks
and 1 C-18 SepPak. The eluting fractions containing radio-
activity were used directly in rodent studies. The identity of
the radiolabeled product was confirmed by comparing the Rf
of the radioactive product visualized with radiometric TLC
with the Rf of the authentic 19F compound visualized with
ninhydrin stain (Rf ) 0.6, Alltech 0.25 mm RP Chiralplates,
20:5:5 CH3CN:H2O:MeOH). In all radiosyntheses, the only
peak present on radiometric TLC analysis corresponded to
5a ,b, and the radiochemical purity of the product exceeded
99%. The isolated radiochemical yields were determined using
a dose calibrator (Capintec CRC-712M).
Am in o Acid Up t a k e In h ib it ion Assa ys. The 9L glio-
sarcoma cells were initially grown as monolayers in T-flasks
containing Dulbecco’s Modified Eagle’s Medium (DMEM)
under humidified incubator conditions (37 °C, 5% CO2/95%
air). The growth media was supplemented with 10% fetal calf
serum and antibiotics (10 000 units/mL penicillin and 10 mg/
mL streptomycin). The growth media was replaced three times
per week, and the cells were passaged so that the cells would
reach confluency in a week’s time.
When the monolayers were confluent, cells were prepared
for experimentation in the following manner. Growth media
was removed from the T-flask, and the monolayer cells were
exposed to 1 X trypsin:EDTA for ∼1 min to weaken the protein
attachments between the cells and the flask. The flask was
then slapped, causing the cells to release. Supplemented media
was added to inhibit the proteolytic action of the trypsin, and
the cells were aspirated through an 18 Ga needle until they
were monodisperse. A sample of the cells was counted under
a microscope using a hemocytometer, and the live/dead fraction
was estimated through trypan blue staining (>98% viability).
The remainder of the cells was placed into a centrifuge tube
and centrifuged at 75G for 5 min, and the supernatant was
removed. The cells were then resuspended in amino acid/
serum-free DMEM salts.
In this study, approximately 4.55 × 105 cells were exposed
to either [18F]5a or [18F]5b (5 µCi) in 3 mL of amino acid free
media ( transporter inhibitors (10 mM) for 30 min under
incubator conditions in 12 mm × 75 mm glass vials. Each
assay condition was performed in duplicate. After incubation,
cells were twice centrifuged (75G for 5 min) and rinsed with
ice-cold amino acid/serum-free DMEM salts to remove residual
activity in the supernatant. The vials were placed in a Packard
Cobra II Auto-Gamma counter, the raw counts were decay-
corrected, and the activity per cell number was determined.
The data from these studies (expressed as percent uptake
relative to control) were graphed using Excel, with statistical
comparisons between the groups analyzed using a one way
ANOVA (GraphPad Prism software package).
mined in 16 normal male Fischer 344 rats (200-250 g) after
intravenous injection of ∼85 µCi of [18F]5a or [18F]5b in 0.3
mL of sterile water. The animals were allowed food and water
ad libitum before the experiment. Following anesthesia in-
duced with an intramuscular injection of 0.1 mL/100 g of a
1:1 ketamine (500 mg/mL):xylazine (20 mg/mL) solution, the
radiolabeled amino acid was injected into the rats via tail vein
catheters. Groups of four rats were killed at 5, 30, 60, and 120
min after injection of the dose. The animals were dissected,
and selected tissues were weighed and counted along with dose
standards in a Packard Cobra II Auto-Gamma Counter. The
raw counts were decay-corrected, and the counts were normal-
ized as the percent of total injected dose per gram of tissue
(%ID/g). A comparison of the uptake of activity in the tissues
at each time point was analyzed using a one way ANOVA
(GraphPad Prism software package).
The tissue distribution of radioactivity was also determined
in tumor-bearing Fischer 344 rats following intravenous
injection of ∼35 µCi of [18F]5a or [18F]5b in 0.3 mL of sterile
water. The procedure was similar to that already described
for normal rats with the following modifications. The tail vein
injections were performed in awake animals using a RTV-190
rodent restraint device (Braintree Scientific) to avoid mortality
accompanying anesthesia in the presence of an intracranial
mass. The animals were killed at 5, 60, or 120 min postinjec-
tion. The same tissues were assayed as in normal rats with
the addition of the tumor tissue, and the corresponding region
of brain contralateral to the tumor was excised and used for
comparison. The uptake in the tumor and contralateral brain
at each time point was compared via a two-tailed t-test for
paired observations (GraphPad Prism software package).
Ack n ow led gm en t. We acknowledge the use of
Shared Instrumentation provided by grants from the
NIH and the NSF for the mass spectroscopy data.
Su p p or tin g In for m a tion Ava ila ble: Mass spectroscopy
data for compound 12b. This material is available free of
charge via the Internet at http://pubs.acs.org.
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