Synthesis, radiolabeling, and biological evaluation of (R)- and (S)-2-amino-3-[18F]fluoro-2-methylpropanoic acid (FAMP) and (R)- and (S)-3-[18F]fluoro-2-methyl-2-n-(methylamino)propanoic acid (NMeFAMP) as potential PET radioligands for imaging brain tumors

Article abstract of DOI:10.1021/jm900556s

The non-natural amino acids (R)- and (S)-2-amino-3-fluoro-2-methylpropanoic acid 5 and (R)- and (S)-3-fluoro-2-methyl-2-N-(methylamino)propanoic acid 8 were synthesized in shorter reaction sequences than in the original report starting from enantiomerically pure (S)- and (R)-R-methyl-serine, respectively. The reaction sequence provided the cyclic sulfamidate precursors for radiosynthesis of (R)- and (S)-[¹⁸F]5 and (R)- and (S)-[ ¹⁸F]8 in fewer steps than in the original report. (R)- and (S)-[ ¹⁸F]5 and(R)-and (S)-[¹⁸F]8 were synthesized by no-carrier-added nucleophilic [¹⁸F]fluorination in 52-66% decay - corrected yields with radiochemical purity over 99%. The cell assays showed that all four compounds were substrates for amino acid transport and enter 9L rat gliosarcoma cells in vitro at least in part by system A amino acid transport. The biodistribution studies demonstrated that in vivo tumor to normal brain ratios for all compounds were high with ratios of 20:1 to115:1 in rats with intracranial 9L tumors. The (R)-enantiomers of [¹⁸F]5 and [ ¹⁸F]8 demonstrated higher tumor uptake in vivo compared to the (S)-enantiomers. 2009 American Chemical Society.

Full text of DOI:10.1021/jm900556s

876 J. Med. Chem. 2010, 53, 876–886
DOI: 10.1021/jm900556s
Synthesis, Radiolabeling, and Biological Evaluation of (R)- and
(S)-2-Amino-3-[18F]Fluoro-2-Methylpropanoic Acid (FAMP) and
(R)- and (S)-3-[18F]Fluoro-2-Methyl-2-N-(Methylamino)propanoic Acid (NMeFAMP)
as Potential PET Radioligands for Imaging Brain Tumors
Weiping Yu,† Jonathan McConathy,§,† Larry Williams,† Vernon M. Camp,† Eugene J. Malveaux,† Zhaobin Zhang,‡
Jeffrey J. Olson,‡ and Mark M. Goodman*,†
†Department of Radiology and ‡Department of Neurosurgery , School of Medicine, Emory University, 1364 Clifton Road NE, Atlanta,
Georgia 30322. § Currently at Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO 63110.
Received April 30, 2009
The non-natural amino acids (R)- and (S)-2-amino-3-fluoro-2-methylpropanoic acid 5 and (R)- and (S)-
3-fluoro-2-methyl-2-N-(methylamino)propanoic acid 8 were synthesized in shorter reaction sequences
than in the original report starting from enantiomerically pure (S)- and (R)-R-methyl-serine, respec-
tively. The reaction sequence provided the cyclic sulfamidate precursors for radiosynthesis of (R)- and
(S)-[18F]5 and (R)- and (S)-[18F]8 in fewer steps than in the original report. (R)- and (S)-[18F]5 and(R)-
and (S)-[18F]8 were synthesized by no-carrier-added nucleophilic [18F]fluorination in 52-66% decay-
corrected yields with radiochemical purity over 99%. The cell assays showed that all four compounds
were substrates for amino acid transport and enter 9L rat gliosarcoma cells in vitro at least in part by
system A amino acid transport. The biodistribution studies demonstrated that in vivo tumor to normal
brain ratios for all compounds were high with ratios of 20:1 to115:1 in rats with intracranial 9L tumors.
The (R)-enantiomers of [18F]5 and [18F]8 demonstrated higher tumor uptake in vivo compared to the
(S)-enantiomers.
Introduction
certain neoplasm, and high physiological uptake in certain
organs and tissues, including the kidneys, urinary tract, and
normal gray matter of the brain.3,4
The development of radiotracers for tumor imaging is a
major focus of radiopharmaceutical research. A number of
classes of compounds that accumulate preferentially in neo-
plastic tissues have been investigated for this purpose, includ-
ing metabolically based radiotracers such as carbohydrates,
aminoacids, and nucleosides. The most widely used metabolic
imaging agent for oncologic applications, 2-[18F]fluoro-2-
deoxy-D-glucose ([18F]FDGa), has demonstrated clinical uti-
lity in staging, treatment planning, and monitoring response
to therapy for a number of types of cancers.1,2 While
[18F]FDG has proven very useful, this radiopharmaceutical
does have some limitations for tumor imaging including high
uptake in inflammatory tissue, low or variable uptake in
Amino acids are important biological substrates in virtually
all biological process and are required nutrients for cell
growth,5-7 and many tumor cells demonstrate increased
amino acid transport relative to normal tissues.8,9 Certain
amino acid transporters also are involved in cell signaling and
may play important roles in tumor biology.10-12 A variety of
radiolabeled amino acids have been developed as potential
tumor imaging agents for positron emission tomography (PET)
and single photon emission tomography (SPECT). Non-natural
amino acids generally have higher metabolic stability than
natural proteogenic amino acids, and this increased stability
can simplify the interpretation of diagnostic images and kinetic
analysis. A number of radiolabeled amino acids including
tyrosine analogues O-(2-[18F]fluoroethyl)-L-tyrosine (FET)13
and 3-[123I]iodo-R-methyl-L-tyrosine (IMT),5,14-16 L-[11C-
methyl]methionine (MET)5,17,18 and anti-1-amino-3-[18F]-
fluorocyclobutane-1-carboxylic acid (FACBC)19 have demon-
strated improved imaging properties relative to [18F]FDG in
human patients with gliomas.
*To whom correspondence should be addressed. Phone: (404) 727-
9366. Fax: (404) 727-4366. E-mail: mgoodma@emory.edu.
a Abbreviations: ACS, alanine-cysteine-serine; AIB, 2-aminoiso-
butyric acid; BBB, blood-brain barrier; BCH, 2-amino-bicyclo-
[2.2.1]heptane-2-carboxylic acid; Boc, tert-butoxycarbonyl; DMB,
4,4’-dimethoxybenzhydryl; DMEM, Dulbecco’s Modified Eagle’s Med-
ium; EDTA, ethylenediaminetetraacetic acid; EOB, end of bombard-
ment; EOS, end of synthesis; FACBC, anti-1-amino-3-fluoro-
cyclobutane-1-carboxylic acid; FAMP, 2-amino-3-fluoro-2-methylpro-
panoic acid; FDG, 2-fluoro-2-deoxy-D-glucose; FET, O-(2-fluoroethyl)-
L-tyrosine; HBSS, Hank’s balanced salt solution; ID, injected dose;
IMT, 3-iodo-R-methyl-L-tyrosine; IVAIB, 2-amino-2-methyl-4-iodo-
3-(E)-butenoic acid; MeAIB, 2-(methylamino)isobutryic acid; MET,
L-methionine; NCA, no-carrier-added; DCY, decay-corrected-yield;
NMeFAMP, 3-fluoro-2-methyl-2-(methylamino)propanoic acid; PET,
positron emission tomography; PBS, phosphate buffer solution;
SPECT, single photon emission tomography.
In this study, we describe the synthesis, radiolabeling, and
biological evaluation of the (S)- and (R)-enantiomers of
2-amino-3-[18F]fluoro-2-methyl propanoic acid (FAMP, 5)
and 3-[18F]fluoro-2-methyl-2-(methylamino)propanoic acid
(NMeFAMP, 8), which are fluorinated analogues of 2-amino-
isobutyric acid (AIB) and 2-(methylamino)isobutryic acid
(MeAIB), respectively. The major rationale for the development
r
pubs.acs.org/jmc
Published on Web 12/22/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 877
of [18F]FAMP and [18F]NMeFAMP was based on preclinical
results obtained with [11C]AIB20 and [11C]MeAIB).21 These
compounds are primarily A-type amino acid transporter
substrates, and racemic mixtures of both [18F]5 and [18F]8
have demonstrated high tumor to brain ratios in the 9L
gliosarcoma rat tumor model, in part as a result of low normal
brain uptake due to the absence of A-type amino acid trans-
port across the luminal surface of normal endothelium of the
blood-brain barrier (BBB).22 Our recent comparisons of the
in vitro and in vivo properties of (S)- and (R)-2-amino-
2-methyl-4-[123I]iodo-3-(E)-butenoic acid ([123I]IVAIB),23
iodovinyl substituted analogues of AIB, demonstrated that
the stereochemistry of IVAIB at the R-carbon strongly affects
both in vitro and in vivo properties. In the 9L gliosarcoma
model, (S)-[123I]IVAIB, an A-type amino acid transporter
substrate, demonstrated superior biological properties com-
pared to the (R)-enantiomer. On the basis of the above
observation and the promising results obtained with racemic
5 and 8, our goal was to improve the synthetic route and to
evaluate the individual (S)- and (R)-enantiomers of 5 and 8 in
the 9L gliosarcoma model to determine the effect of stereo-
chemistry on the biological properties of these compounds.
the synthesis of nonradioactive (R)- and (S)-5 and (R)- and
(S)-8, as well as radiolabeling precursors (S)- and (R)-7 for
(R)- and (S)-[18F]8 (Scheme 1).
The preparation of (R)- and (S)-8 from the cyclic sulfami-
date intermediates (S)- and (R)-4, respectively, required
selective removal of the N-Boc group. Attempts to remove
the N-Boc group in the presence of the tert-butyl ester using
p-toluenesulfonic acid22 or with anhydrous hydrochloric
acid in dioxane28 were not successful, presumably due to
high sensitivity of the cyclic sulfamidate toward the acidic
condition. Selective removal of the N-Boc group was
achieved by treatment of 4 with methanesulfonic acid in
tert-butyl acetate/dichloromethane as solvent.29 The success
of this reaction is likely because the removal of Boc group is
an irreversible process due to protonation of the amine group
and loss of CO2. In contrast, the acidic deprotection of the
tert-butyl ester is a reversible process due to the nature of
the tert-butyl carbonium ion mediated equilibrium with a
tert-Buþ source. The likely reaction route is displayed in
Scheme 2. The possible byproducts or intermediates 9 and 10
and their interchanging between 4 and 6 were observed by
TLC at different reaction points. Using a mild organic acid
such as methanesulfonic acid under anhydrous condition
to prevent cyclic sulfamidate ring-opening was the key to
successfully prepare (S)- and (R)-6 from (S)-4 and (R)-4,
respectively. The N-methylation of (S)-6 and (R)-6 was
achieved by utilizing dimethyl sulfate with sodium hydride,30
which provided (S)-7 and (R)-7 in good yields. Initial
attempts using methyl iodide as the methylating agent for
this reaction were unsuccessful.
Radiochemistry. The radiosyntheses of (R)-[18F]5,
(S)-[18F]5, (R)-[18F]8, and (S)-[18F]8 were carried out
as shown in Scheme 3 with no-carrier-added (NCA)
cyclotron-produced [18F]fluoride using a modified auto-
mated method as previously described.31 The cyclic sul-
famidate precursors (S)-4 and (R)-4 provided an average
52 ( 12% of (R)-[18F]5 (n = 10) and 56 ( 12% of (S)-[18F]5
(n = 6) decay corrected yields (DCY), respectively, in over
99% radiochemical purity based on radiometric TLC.
Similarly, treatment of (S)-7 and (R)-7 under the same
conditions gave an average 66 ( 12% of (R)-[18F]8 (n =
10) and 66 ( 18% of (S)-[18F]8 (n = 8) DCY, respectively,
in over 99% radiochemical purity. Unreacted [18F]-
fluoride, radiolabeled ionic byproducts as well as hydro-
gen chloride from the hydrolysis step were removed by
passing the reaction mixture through an ion-retardation
resin column followed by an alumina N Sep-Pak. Less
polar and organic byproducts were removed by using a
HLB Oasis reverse-phase cartridge. The purified final
products (R)-[18F]5, (S)-[18F]5, (R)-[18F]8, and (S)-[18F]8
were obtained in pH 6-7 normal saline solution and used
directly in the cell assays and the rodent studies. In a
representative synthesis, a total of 155 mCi of (R)-[18F]5 at
end of synthesis (EOS) was obtained from 387 mCi of
[18F]fluoride at end of bombardment (EOB) in a synthesis
time of approximately 85 min using 1 mg (3 μmol) of cyclic
sulfamidate precursor (S)-4.
Results and Discussion
Chemistry. The nonradioactive amino acids (S)- and (R)-5
were prepared in a simple five-step synthesis as shown in
Scheme 1. This method is an improvement over the originally
reported method which employed a Strecker-type reaction to
prepare compound 5 as a racemic mixture.22 Commercially
available (S)- and (R)-R-methyl-serine were treated with di-
tert-butyl dicarbonate to give carbamates (S)- and (R)-1,
which were used without purification in the next reaction.
Because of the presence of both carboxylic acid and hydroxy
functionalities, protection of the carboxylic function as a
tert-butyl ester without ether formation was necessary. The
use of tert-butyl-2,2,2-trichloroacetimidate led to the forma-
tion of both the tert-butyl ester as well as the tert-butyl ether.
In contrast, N,N-dimethylformamide di-tert-butyl acetal24,25
provided selective esterification of the carboxylic acid func-
tion when heated with (S)- or (R)-1 in anhydrous toluene.
The next key step for making the target radiolabeling
precursors (S)- and (R)-4 was preparation of cyclic sulfami-
dites from 2. The typical approach of reacting the N-tert-
butoxycarbonyl (Boc) protected amino alcohols (S)- and
(R)-2 with thionyl chloride in the presence of triethylamine
in low polarity solvent at low temperature failed to give the
corresponding cyclic sulfamidites. Our previous strategy was
to replace the Boc group with the electron releasing group
bis(4-methoxyphenyl)methyl, also known as 4,40-dimethox-
ybenzhydryl (DMB) group.22 While effective, this approach
added at least two synthetic steps to the sequence and
required the preparation of bis(4-methoxyphenyl)chloro-
methane26 for N-alkylation. This problem with cyclization
of the N-Boc compound was successfully solved by utilizing
Posakony’s method,27 which uses a more polar solvent for
cyclization. The carbamate nitrogens of (S)- and (R)-2 are
predicted to be less nucleophilic than their N-alkyl counter-
parts, and the use of acetonitrile allowed formation of the
N-Boc protected cyclic sulfamidites (S)- and (R)-3. Further
oxidation of 3 using sodium periodate with ruthenium(III)
chloride catalysis provided the cyclic sulfamidates (S)-
and (R)-4, which served as radiolabeling precursors for
(R)- and (S)-[18F]5, respectively, and as intermediates for
While the specific activities of (R)- and (S)-[18F]5 and (R)-
and (S)-[18F]8 were not determined directly, the maximum
amount of unlabeled material in the final product arising
from the precursors is about 1 mg in each case. On the basis
of a 150 mCi yield at EOS, the amount of nonradioactive
material in the final dose arising from the precursor is
approximately 7 μg per mCi. This amount is comparable to

878 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Yu et al.
Scheme 1. Syntheses of Labeling Precursors (S)- and (R)-4 and (S)- and (R)-7, and Syntheses of the Fluorine-19 amino Acids (R)- and
(S)-5 and (R)- and (S)-8a
a Reagents and conditions: (a) (Boc)2O, MeOH-Et3N-NaOH (aq), rt. (b) (CH3)2NCH[OC(CH3)3]2, 80-90 °C. (c) SOCl2, Pyr, -40 °C. (d) NaIO4,
RuCl3, CH3CN/H2O, 0 °C to rt. (e) n-Bu4NF, rt, then 3N HCl, 85 °C. (f) MeSO3H, t-BuOAc, DCM, rt. (g) Me2SO4, NaH, THF, rt.
Scheme 2. Possible Route for Deprotection of the Boc Group
at the Presence of the tert-Butyl Ester
Scheme 3. Radiosynthesis of (R)- and (S)-[18F]5 and (R)- and
(S)-[18F]8
these transport systems are sodium-dependent except system
L. Prior studies demonstrate that AIB and its analogues
including MeAIB, (R,S)-[18F]5, and (R,S)-[18F]8 enter cells
mainly through system A transport.20,22 To determine the
transport mechanisms involved in the uptake of (S)- and
(R)-[18F]5 and (S)- and (R)-[18F]8 by 9L gliosarcoma tumor
cells, inhibition assays were performed using cultured rat 9L
gliosarcoma cells in the presence and absence of amino acid
carrier inhibitors. MeAIB is a selective competitive inhibitor
of system A transport, and 2-amino-bicyclo[2.2.1]heptane-
2-carboxylic acid (BCH) is an inhibitor of system L transport
although it is not entirely selective for system L in the
presence of sodium.35,38,39 The combination of 10 mM
alanine-cysteine-serine (ACS, 3.3 mM of each amino acid)
was also used for uptake inhibition as these amino acids are
substrates for system ASC. The uptake data was normalized
and expressed as mean percent uptake relative to the control
condition (Table 1). In all cases, ACS inhibited more than
half of the uptake compared to control (58.2-90.0%). In the
case of (R)- and (S)-[18F]5, both BCH and MeAIB inhibited
uptake to certain degrees, with 67-75% inhibition by BCH,
and 31-58% by MeAIB. In the case of (R)- and (S)-[18F]8,
MeAIB inhibited 77-92% of uptake, whereas BCH inhib-
ited 28-54% of uptake relative to controls (Figure 1).
On the basis of the results of these inhibition studies, (R)-
and (S)-[18F]5 enter 9L gliosarcoma cells in vitro involves
both system A and nonsystem A transport, likely system L.
the amount of nonradioactive material present in doses of
[18F]FDG, which arises from the triflate precursor.32 Simi-
larly, the non-natural amino acid anti-[18F]FACBC contains
nonradioactive material arising from its triflate precursor.31
On the basis of the average dosage of 5-20 μCi of [18F]5 or
[18F]8 per animal in the in vivo study, the maximum amount
of unlabeled material arising from the precursor associated
with (R)- and (S)-[18F]5 and (R)- and (S)-[18F]8 per injection
was approximately 0.034-0.13 μg. The major byproducts
arising from the labeling precursor are expected to consist of
R-methyl-serine or N-methyl-R-methyl-serine arising from
opening of C-5 position of cyclic sulfamidate precursors by
water or hydroxide ions.
Cell Uptake Assays. Amino acids enter normal and neo-
plastic cells through carrier-mediated uptake from the ex-
tracellular fluid. For radiolabeled amino acids, tumor
uptake is primarily determined by amino acid transport
rather than protein synthesis.5,6 The most abundant amino
acid transport systems in most mammalian cells are system
A, system L, system ASC, and system Gly.6,12,33-37 All of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 879
Table 1. 9L Cell Uptake of (R)- and (S)-[18F]5 and (R)- and (S)-[18F]8 at 30 min Incubation with or without Inhibitorsa
control
BCH
MeAIB
ACS
(R)-FAMP 5
uptake
inhibition
p
13.06 ( 0.23
4.27 ( 0.21
67
<0.0001
5.51 ( 0.16
58
<0.0001
1.70 ( 0.02
87
<0.0001
(S)-FAMP 5
uptake
inhibition
p
4.77 ( 0.73
7.08 ( 0.47
3.87 ( 0.22
1.21 ( 0.13
75
<0.002
3.29 ( 0.15
31
<0.03
1.33 ( 0.27
72
<0.002
(R)-NMeFAMP 8
(S)-NMeFAMP 8
uptake
inhibition
p
3.26 ( 0.22
54
<0.0001
0.60 ( 0.12
92
<0.0001
0.75 ( 0.09
89
<0.0001
uptake
inhibition
p
2.76 ( 0.20
29
<0.002
0.82 ( 0.11
79
<0.0001
1.62 ( 0.08
58
<0.0001
a Values are reported as percent uptake of the initial dose per 0.5 million cells (% ID/5 ꢀ 105 cells) (standard deviation (SD) (n= 3). Inhibition is expressed as
percent relative to control (%). p values represent comparisons of uptake with inhibitors to control uptake for each radiotracer using one-way ANOVA tests.
Figure 1. 9L Cell percent uptake of (R)-[18F]5, (S)-[18F]5, (R)-[18F]8, and (S)-[18F]8 relative to control.
(R)- and (S)-[18F]8 are relatively specific substrates for
system A transport but the inhibition of uptake of these
compounds in the presence of BCH, especially for (R)-8,
suggests that compounds (R)- and (S)-[18F]8 entered 9L
gliosarcoma cells in vitro through nonsystem A transport
as well. Given the very low normal brain uptake in the
biodistribution studies, it was unclear if the nonsystem A
transport likely attributable to system L observed in vitro
made a significant contribution to in vivo uptake.
Biodistribution Studies in Rats with Intracranial 9L Glio-
sarcoma Tumors. Uptake in Tumors and in Normal Brain
Tissues. Prior studies using radiolabeled amino acids includ-
ing racemic [18F]5 and [18F]8 in the 9L gliosarcoma tumor
model demonstrated similar tissue distributions of radio-
activity in tumor-bearing and normal rats.19,22,38,39 For this
reason, biodistribution was measured only in rats with
intracranial tumors. The results of the tumor and normal
brain uptake of (R)-[18F] 5, (S)-[18F]5, (R)-[18F]8, and
(S)-[18F]8 are summarized in Table 2. The uptake of radio-
activity by tumors was significantly higher than in normal
brain tissue at each time point for all four compounds (p <
0.002). Tumor uptake of radioactivity for (R)-[18F]5 was
2.4-2.8% injected dose per gram of tissue (%ID/g). Under
the same conditions, tumor uptake of radioactivity for
(S)-[18F]5 was lower, in the range of 1.4-2.1%ID/g. While
the uptake in normal brain contralateral tissue was similar
for both [18F]5, the ratios of tumor to normal brain uptake
for (R)-[18F]5 was slightly higher than (S)-[18F]5.
Tumor uptake of (R)-[18F]8 was accumulated from 1.5 to
2.8%ID/g over the time course of 120 min. Tumor uptake of
(S)-8 was lower and stable around 1.5%ID/g under the same
conditions. The brain uptake ranged from 0.02 to 0.06%ID/g
at all time points for both [18F]8, which resulted in the tumor to
brain ratios of (R)-[18F]8 much higher than that of (S)-[18F]8.

880 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Yu et al.
Table 2. Uptake of Radioactivity in Tumor and Brain of Tumor-Bearing Fischer Rats Following Intravenous Administration of Radiotracers [18F]5
and [18F]8a
radiotracer
15 min
30 min
60 min
120 min
(R)-[18F]5
brain
tumor
tumor:brain ratio
0.06 ( 0.01
2.46 ( 0.13
38.2
0.10 ( 0.02
2.82 ( 0.32
28.6
0.09 ( 0.02
2.42 ( 0.49
27.5
(S)-[18F]5
(R)-[18F]8
(S)-[18F]8
brain
tumor
tumor:brain ratio
0.07 ( 0.005
1.38 ( 0.06
21.1
0.06 ( 0.005
1.82 ( 0.10
30.9
0.08 ( 0.02
2.13 ( 0.23
27.7
brain
tumor
tumor:brain ratio
0.06 ( 0.008
1.46 ( 0.24
25.0
0.04 ( 0.01
2.18 ( 0.11
53.2
0.05 ( 0.02
2.64 ( 0.62
58.2
0.02 ( 0.01*
2.78 ( 1.13*
115.4
brain
tumor
tumor:brain ratio
0.06 ( 0.02
1.29 ( 0.16
20.4
0.06 ( 0.02
1.77 ( 0.32
30.2
0.05 ( 0.01
1.46 ( 0.30
28.7
0.04 ( 0.01**
1.35 ( 0.42**
35.4
a Values are reported as mean percent injected dose per gram tissue (%ID/g) ( standard deviation; n = 5 ((R)-[18F]5, (R)-[18F]8), and n = 4
((S)-[18F]5, (S)-[18F]8) at each time point. p values were calculated using 1-way ANOVA tests (tumor vs. brain); *p e 0.0006; **p < 0.002; all other cases
p < 0.0001.
Figure 2. Comparisons of tumor uptake (%ID/g) in 9L tumor-bearing rats for (R)-[18F]5, (S)-[18F]5, (R)-[18F]8, and (S)-[18F]8.
These results in 9L gliosarcoma tumors showed that the
activity accumulated to high levels in the tumors by 30 min
and stayed at similar high levels through the 120 min study
course for all four amino acids. The tumor uptake of these
four compounds in 9L tumor-bearing rats is depicted in
Figure 2. Statistically significant differences in tumor uptake
were observed between the four amino acids in the biodis-
tribution studies: (R)-[18F]5 > (S)-[18F]5 at 30 and 60 min
(p e 0.02), (R)-[18F]5 > (R)-[18F]8 at 30 min (p < 0.03),
(R)-[18F]5 > (S)-[18F]8 at all time points (p e 0.02),
(R)-[18F]8 > (S)-[18F]5 at 30 min (p e 0.001), (S)-[18F]5 >
(S)-[18F]8 at 120 min (p < 0.03), (R)-[18F]8 > (S)-[18F]8 at 60
min (p e 0.01).
The normal brain uptake of these four compounds in 9L
tumor-bearing rats is presented in Figure 3. There were
statistically significant differences in the levels of normal
brain uptake for these amino acids at the following time
points (p < 0.04 in all cases): (R)-[18F]5 > (S)-[18F]5 at 60
min, (R)-[18F]5 > (R)-[18F]8 at all time points, (R)-[18F]5 >
(S)-[18F]8 at 60 and 120 min, (S)-[18F]5 > (R)-[18F]8 at
30 and 120 min, (S)-[18F]8 > (R)-[18F]8 at 120 min.
The tumor and brain uptake observed with (R)- and
(S)-[18F]5 demonstrate that the stereochemistry of the
R-carbon affects the in vivo biological behavior of these
compounds. The (R)-enantiomer of [18F]5 demonstrated
statistically significant higher tumor uptake than the (S)-
enantiomer at the 30 and 60 min time points while the normal
brain uptake with (R)-[18F]5 was statistically significantly
The high tumor to brain ratios observed with these amino
acids was in part due to the low normal brain uptake as well.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 881
Figure 3. Comparisons of normal brain uptake (%ID/g) in 9L tumor-bearing rats for (R)-[18F]5, (S)-[18F]5, (R)-[18F]8, and (S)-[18F]8.
higher than with (S)-enantiomer at 60 min but not at 30 or
120 min. In the case of (R)- and (S)-[18F]8, the (R)-enantio-
mer showed statistically significant higher tumor uptake at
60 min than the (S)-enantiomer. The tumor uptake with
(R)-[18F]8 at 120 min was not statistically significantly
different than the (S)-enantiomer despite the large difference
in average uptake values, which is likely due to variability
in the %ID/g values at this time point. The uptake in
normal brain with both enantiomers of [18F]8 was signifi-
cantly different at 120 min but not at the other time
points evaluated. These results are similar to (R)- and
(S)-[123I]IVAIB, although the difference between the enan-
tiomers of 5 and 8 in the 9L model are not as large as for
Table 3. Biodistribution of Radioactivity in Tissues (Other than Tumor
and Brain) of Tumor-Bearing Fischer Rats Following Intravenous
Administration of (R)-[18F]5a
tissue
30 min
60 min
120 min
blood
heart
lung
0.28 ( 0.03
0.32 ( 0.05
0.37 ( 0.08
0.60 ( 0.06
2.96 ( 0.52
0.72 ( 0.07
3.47 ( 0.69
0.32 ( 0.05
0.23 ( 0.10
0.15 ( 0.02
0.25 ( 0.01
0.34 ( 0.05
0.40 ( 0.03
0.65 ( 0.11
2.05 ( 0.47
0.71 ( 0.07
2.96 ( 0.46
0.52 ( 0.07
0.36 ( 0.12
0.17 ( 0.01
0.16 ( 0.03
0.23 ( 0.04
0.23 ( 0.03
0.32 ( 0.07
1.17 ( 0.17
0.39 ( 0.05
2.09 ( 0.20
0.36 ( 0.09
0.26 ( 0.02
0.16 ( 0.03
liver
pancreas
spleen
kidney
muscle
bone
testis
[
123I]IVAIB.23
a Values are reported as mean percent injected dose per gram tissue
(%ID/g) ( standard deviation; n = 5 at each time point.
Low brain uptake observed with all four amino acids is
compatible with primarily system A transport in vivo, which
is absent at the luminal surface of the endothelium of the
normal BBB. The low brain uptake of (R)- and (S)-[18F]5 and
(R)- and (S)-[18F]8 is consistent with the earlier studies
performed with [11C]AIB20 as well as racemic [18F]5 and
[18F]8.22 The in vitro inhibition of uptake of these com-
pounds by BCH indicates nonsystem A transport and is
compatible with a component of system L transport. Because
system L transport occurs across the normal BBB, substrates
for system L would be expected to show uptake in normal
brain. The in vitro assay conditions were performed in the
absence of amino acids, and it is possible that endogenous
system L substrates competitively inhibit the transport of
(R)- and (S)-[18F]5 and (R)- and (S)-[18F]8 in vivo. Future
imaging studies in nonhuman primates and in human cancer
patients will provide more detailed information regarding
the biodistribution and kinetics of tracers in normal and
neoplastic tissues.
[18F]5 and [18F]8.19,22 The activity uptake of [18F]FDG at 60
min post injection was 1.05%ID/g in tumor and 1.30%ID/g
in normal brain, giving a tumor to brain ratio of 0.8:1. This
low ratio is consistent with the high physiologic uptake of
[18F]FDG in normal brain tissue. For anti-[18F]FACBC, the
uptake ratio of tumor to brain at 60 min post injection was
6.6:1, with 1.72%ID/g in tumor and 0.26%ID/g in brain
tissue. The higher tumor to brain ratios observed with
(R)-[18F]8 and (R)- and (S)-[18F]5 compared to [18F]FDG
and anti-[18F]FACBC were due to a combination of higher
levels of tumor uptake of tracer and much lower uptake in
normal brain. The higher tumor to brain ratio of compound
(S)-[18F]8 compared to [18F]FDG and anti-[18F]FACBC is
due to the lower normal brain uptake.
Uptake in Normal Tissues. The results of the biodistribu-
tion studies with (R)- and (S)-[18F]5 in normal tissues of
tumor-bearing rats are shown in Tables 3 and 4. The organs
which received highest radioactivity were kidneys and pan-
creas ( p < 0.0001 compared to other tissues studied), with
(S)-[18F]5 higher than (R)-[18F]5 at all time points. The
activity in these tissues remained above the activity in other
For all four compounds studied, the tumor to brain ratios
are higher than those reported for [18F]FDG and anti-
[18F]FACBC in the same 9L gliosarcoma tumor model and
similar to the results obtained with the racemic mixtures of

882 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Yu et al.
Table 4. Biodistribution of Radioactivity in Tissues (Other than Tumor
and Brain) of Tumor-Bearing Fischer Rats Following Intravenous
Administration of (S)-[18F]5a
Table 6. Biodistribution of Radioactivity in Tissues (Other than Tumor
and Brain) of Tumor-Bearing Fischer Rats Following Intravenous
Administration of (S)-[18F]8a
tissue
30 min
60 min
120 min
tissue
15 min
30 min
60 min
120 min
blood
heart
lung
0.58 ( 0.04
0.43 ( 0.05
0.67 ( 0.09
0.61 ( 0.07
3.75 ( 0.66
0.73 ( 0.07
8.71 ( 0.76
0.47 ( 0.02
0.30 ( 0.09
0.19 ( 0.02
0.40 ( 0.03
0.41 ( 0.06
0.47 ( 0.07
0.54 ( 0.07
3.91 ( 0.49
0.62 ( 0.04
7.62 ( 0.39
0.37 ( 0.03
0.30 ( 0.04
0.16 ( 0.01
0.32 ( 0.04
0.34 ( 0.02
0.36 ( 0.02
0.42 ( 0.04
2.79 ( 0.49
0.51 ( 0.02
6.88 ( 0.83
0.36 ( 0.06
0.31 ( 0.02
0.17 ( 0.01
blood
heart
lung
0.54 ( 0.02
0.32 ( 0.02
0.86 ( 0.10
0.83 ( 0.06
3.52 ( 1.15
0.57 ( 0.10
11.58 ( 1.88
0.25 ( 0.02
0.46 ( 0.07
0.19 ( 0.01
0.31 ( 0.03
0.30 ( 0.06
0.87 ( 0.20
0.71 ( 0.01
3.40 ( 0.38
0.51 ( 0.05
9.48 ( 2.48
0.26 ( 0.07
0.32 ( 0.07
0.13 ( 0.01
0.24 ( 0.02
0.31 ( 0.04
0.50 ( 0.05
0.88 ( 0.29
4.34 ( 1.13
0.53 ( 0.06
5.18 ( 0.56
0.24 ( 0.03
0.37 ( 0.03
0.13 ( 0.03
0.14 ( 0.01
0.21 ( 0.03
0.42 ( 0.16
0.60 ( 0.12
3.78 ( 0.39
0.49 ( 0.05
2.22 ( 0.16
0.22 ( 0.03
0.25 ( 0.06
0.08 ( 0.01
liver
liver
pancreas
spleen
kidney
muscle
bone
pancreas
spleen
kidney
muscle
bone
testis
testis
a Values are reported as mean percent injected dose per gram tissue
a Values are reported as mean percent injected dose per gram tissue
(%ID/g) ( standard deviation; n = 4 at each time point.
(%ID/g) ( standard deviation; n = 4 at each time point.
uptake in brain of less than 0.07%ID/g at all time points (p <
0.0001 compared to other tissues studied).
Table 5. Biodistribution of Radioactivity in Tissues (Other than Tumor
and Brain) of Tumor-Bearing Fischer Rats Following Intravenous
Administration of (R)-[18F]8a
The high pancreatic and renal uptake in rodent models has
been reported for numerous radiolabeled amino acids in-
cluding substrates for system L and system A.21-23,38-47 The
lack of significant accumulation of radioactivity in bone
indicates that significant in vivo defluorination resulting
from metabolism did not occur with either of these com-
pounds during the two hour studies. Overall, the (R)-enan-
tiomers of 5 and 8 have better imaging properties in the 9L
model than the corresponding (S)-enantiomers with lower
uptake in the kidneys and pancreas. This difference may be
due to the similarity of the R-carbon stereochemistry of
(R)-[18F]5 and (R)-[18F]8 to the naturally occurring amino
acids (S)-serine and (S)-alanine.
tissue
15 min
30 min
60 min
120 min
blood
heart
lung
0.41 ( 0.03
0.55 ( 0.14
0.79 ( 0.28
0.60 ( 0.12
2.25 ( 1.16
0.57 ( 0.06
9.22 ( 1.26
0.27 ( 0.07
0.51 ( 0.09
0.19 ( 0.07
0.32 ( 0.04
0.23 ( 0.02
0.36 ( 0.02
0.61 ( 0.11
2.72 ( 0.62
0.63 ( 0.06
8.91 ( 1.32
0.23 ( 0.03
0.48 ( 0.08
0.15 ( 0.01
0.24 ( 0.08
0.24 ( 0.08
0.38 ( 0.19
0.52 ( 0.07
3.41 ( 0.48
0.59 ( 0.11
4.44 ( 0.89
0.20 ( 0.02
0.51 ( 0.08
0.15 ( 0.06
0.14 ( 0.04
0.22 ( 0.01
0.25 ( 0.06
0.40 ( 0.09
3.39 ( 0.79
0.62 ( 0.09
2.45 ( 1.10
0.22 ( 0.03
0.44 ( 0.13
0.16 ( 0.06
liver
pancreas
spleen
kidney
muscle
bone
testis
a Values are reported as mean percent injected dose per gram tissue
(%ID/g) ( standard deviation; n = 5 at each time point.
Conclusions
tissues (p e 0.0003 at all time points). For (R)-[18F]5, the
radioactivity uptake in kidneys and pancreas were 3.5 and
3.0%ID/g at 30 min post injection, respectively, which
decreased to 2.1 and 1.2%ID/g at 120 min post injection,
respectively. In the case of (S)-[18F]5, the uptake of activity in
these tissues were 8.7 and 3.8%ID/g at 30 min post injection,
respectively, which decreased to 6.9 and 2.8%ID/g at
120 min post injection, respectively. With both enantiomers
of [18F]5, the tissues studied including blood, liver, heart,
lung, spleen, bone, muscle, and testis showed relatively low
uptake of radioactivity at 30 min (e0.73% ID/g) which
decreased over the course of the two hour study. The brain
showed the lowest uptake of radioactivity for both enantio-
mers of [18F]5 with less than 0.1%ID/g at all time points (p e
0.0001 compared to other tissues studied).
The current study demonstrates that (R)- and (S)-[18F]5
and (R)- and (S)-[18F]8 can be produced in high radiochemical
yield (>52% DCY) and high radiochemical purity from
stable precursors using a semiautomated synthesis system.
Additionally, a new synthetic strategy provides a more effi-
cient route to enantiomerically pure radiolabeling precursors.
We found that the stereochemistry influenced the uptake of
these compounds by 9L gliosarcoma cells. The inhibition
experiments showed that(R)- and (S)-[18F]5 entered9L tumor
cells in vitro via mixed system A and system L amino acid
transport, with more L-type propensity for (S)-[18F]5. Both
(S)- and (R)-[18F]8 enter 9L cells in vitro primarily via system
A transport. Biodistribution studies with all four amino acids
demonstrated rapid and persistent retention of radioactivity
in rodent brain tumors with excellent tumor to background
ratios of 20:1 to115:1. The stereochemistry at the R-carbon
influenced the in vivo behavior of these compounds, with the
(R)-enantiomers of [18F]5 and [18F]8 leading to significantly
higher tumor uptake than the corresponding (S)-enantiomers
at several time points. The relatively low uptake of radio-
activity of these compounds in most normal tissues with the
exception of the kidney and pancreas suggests that these
compounds might be suitable for imaging extracranial tumors
as well.
For (R)- and (S)-[18F]8, the pattern of biodistribution in
normal tissues of tumor-bearing rats was similar to that of
(R)- and (S)-[18F]FAMP 5. These results are depicted in
Tables 5 and 6. For both enantiomers of [18F]8, the highest
radioactivity uptake occurred in kidneys (p < 0.0001 com-
pared to other tissues studied), which decreased through the
course of study from 9.2%ID/g at 15 min post injection to
2.5%ID/g at 120 min post injection for (R)-[18F]8 and from
11.6%ID/g at 15 min post injection to 2.2%ID/g at 120 min
post injection for (S)-[18F]8. The second highest radioactivity
uptake was found in the pancreas (p < 0.0001 compared to
other tissues studied), which stayed at the same level through
the time course of the study with the average value of 2.9 and
3.8%ID/g for (R)-[18F]8 and (S)-[18F]8, respectively. Similar
to [18F]5, both enantiomers of [18F]8 showed relatively low
uptake in the other organs and tissues, and the lowest activity
Experimental Section
Materials and Instrumentation. All chemicals used were ob-
tained from commercially available sources, were of analytical
or higher grade, and were used without further purification.
Solvents used in reactions and purifications were purchased

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 883
from Aldrich Chemicals Co. (Milwaukee, WI) and from VWR
Scientific Products (West Chester, PA). Thin-layer chromato-
graphy (TLC) analyses were performed with 250 μm thick
layers of fluorescence UV254 silica gel backing on aluminum
plates purchased from Whatman Ltd. (Maidstone, Kent,
England). Flash chromatography was carried out using Merck
Kieselgel silica gel 60 (230-400 mesh). Melting points were
measured in capillary tubes using a Mel-Temp II apparatus
(Laboratory Devices, Inc., Holliston, MA) and are uncor-
(S)- and (R)-2-N-(tert-Butoxycarbonyl)amino-2-methyl-3-
hydroxy-propanoic Acid tert-Butyl Ester (2). To a suspension
of (S)- or (R)-1 (595 mg, 2.72 mmol) in 15 mL dry toluene was
added N,N-dimethylformamide di-tert-butyl acetal (2762 mg,
13.9 mmol) over 10 min at room temperature under an argon
atmosphere. The mixture was heated to reflux at 80-90 °C for
2 h and then at room temperature overnight. The reaction was
washed with water (1 ꢀ 8 mL), saturated sodium bicarbonate
(1 ꢀ 5 mL), and brine (1 ꢀ 10 mL) successively. After drying
over sodium sulfate, the solvent was removed under reduced
pressure. The crude product was purified by flash chromato-
graphy (20% ethyl acetate in hexane) to give (S)- or (R)-2 as
white solid. (S)-2-N-(tert-Butoxycarbonyl)amino-2-methyl-3-
hydroxy-propanoic acid tert-butyl ester ((S)-2), 267 mg (35.7%),
mp 72-74 °C (ethyl acetate/hexane). 1H NMR (CDCl3) δ 1.435
(3H, s), 1.440 (9H, s), 1.475 (9H, s), 3.249 (1H, broad s), 3.707,
3.735 (1H, d, J = 11.2 Hz), 3.986, 4.014 (1H, d, J = 11.2 Hz), 5.32
(1H, broad s). Anal. (C13H25NO5) C, H, N. (R)-2-N-(tert-butox-
ycarbonyl)amino-2-methyl-3-hydroxy-propanoic acid tert-butyl
ester ((R)-2), 290 mg (38.9%), mp 72-74 °C (ethyl acetate/
1
rected. H NMR spectra were recorded on Varian 600, 400,
or 300 MHz spectrometers at NMR Center in Emory Uni-
versity, and chemical shifts (δ values) were reported as parts
per million (ppm) downfield from tetramethylsilane (TMS).
Elemental analyses were performed by Atlantic Microlabs,
Inc. (Norcross, GA) and were within (0.4% of the theoretical
values unless otherwise indicated. A few of the products were
oils which contained trace amounts of solvents and were
characterized by high resolution mass spectrometry in con-
junction with NMR and TLC to confirm identity. In each
and every case, the purity of the identified compound
was greater than 95%. Mass spectrometry was performed
with a JEOL JMS-SX102/SX102A/E or VG 70-S double
focusing mass spectrometer at the Mass Spectrometry Center
at Emory University using high resolution electrospray ioniza-
tion (ESI). Observed masses were within 9 ppm of calculated
values.
1
hexane). H NMR (CDCl3) δ 1.44 (3H, s), 1.45 (9H, s), 1.48
(9H, s), 3.71, 3.74 (1H, d, J = 11.4 Hz), 3.98, 4.02 (1H, d,
J = 11.4 Hz), 5.33 (1H, broad s). Anal. (C13H25NO5) C, H, N.
(S)- and (R)-3-(tert-Butoxycarbonyl)-4-methyl-1,2,3-oxathia-
zolidine-4-carboxylic Acid tert-butyl Ester 2-Oxide (3). To a
solution of thionyl chloride (297 mg, 2.5 mmol) in 4 mL of
anhydrous acetonitrile cooled to -40 °C was added the amino
alcohol (S)- or (R)-2 (275 mg, 1.0 mmol) in 1 mL of anhydrous
acetonitrile under an argon atmosphere followed by the addi-
tion of pyridine (396 mg, 5 mmol). After 15 min, the cold bath
was removed, and the reaction was continued for 10 min. The
solvent was removed under reduced pressure. The crude product
was subjected to silica gel column chromatography (20% ethyl
acetate in hexane) to afford a ∼1.2:1 mixture of cyclic sulfami-
dite diastereomers (S)- or (R)-3 as colorless oil. The purified
mixture of sulfamidite diastereomers was used in the next step
reaction without separation. (S)-3-(tert-Butoxycarbonyl)-
4-methyl-1,2,3-oxathiazolidine-4-carboxylic acid tert-butyl es-
ter 2-oxide ((S)-3), 306 mg (95.3%). 1H NMR (CDCl3) for major
diastereomer: δ 1.49 (9H, s), 1.54 (9H, s), 1.59 (3H, s), 4.57 (1H,
d, J = 8.8 Hz), 4.74 (1H, d, J = 8.8 Hz). 1H NMR (CDCl3) for
minor diastereomer: δ 1.49 (9H, s), 1.55 (9H, s), 1.61 (3H, s),
4.44 (1H, d, J = 8.0 Hz), 5.10 (1H, d, J = 8.0 Hz). Anal. for
mixture of diastereomers (C13H23NO6S), C, H, N. (R)-3-(tert-
Butoxycarbonyl)-4-methyl-1,2,3-oxathiazolidine-4-carboxylic
acid tert-butyl ester 2-oxide ((R)-3), 216 mg (67.2%). 1H NMR
(CDCl3) for major diastereomer: δ 1.47 (9H, s), 1.53 (9H, s), 1.59
(3H, s), 4.55 (1H, d, J = 9.2 Hz), 4.73 (1H, d, J = 8.8 Hz). 1H
NMR (CDCl3) for minor diastereomer: δ 1.49 (9H, s), 1.55 (9H,
s), 1.60 (3H, s), 4.43, 4.45 (1H, d, J = 8.0 Hz), 5.09, 5.11 (1H, d,
J = 8.0 Hz). Anal. for mixture of diastereomers (C13H23NO6S),
C, H, N.
(S)- and (R)-3-(tert-Butoxycarbonyl)-4-methyl-1,2,3-oxathia-
zolidine-4-carboxylic Acid tert-Butyl Ester 2,2-Dioxide (4). A
solution of the diastereomeric sulfamidites (S)- or (R)-3 (154 mg,
0.48 mmol) in 6 mL of acetonitrile was cooled to 0 °C and treated
successively with a catalytic portion of RuCl3 (2 mg, 0.01 mmol)
and sodium periodate (123 mg, 0.58 mmol). The reaction
mixture was then treated with 6 mL of water and stirred at
0 °C for 15 min and then at room temperature overnight.
Following dilution of the reaction mixture with ether (20 mL),
two phases were separated. The aqueous phase was extracted
with ether (2 ꢀ 20 mL). The combined organic layers were
washed with saturated sodium bicarbonate (1 ꢀ 20 mL) and
brine (1 ꢀ 20 mL) and dried over sodium sulfate. The crude
compound was purified on silica gel eluted with 20% ethyl
acetate in hexane to give (S)- or (R)-4 as white solid. (S)-3-(tert-
Butoxycarbonyl)-4-methyl-1,2,3-oxathiazolidine-4-carboxylic
acid tert-butyl ester 2,2-dioxide ((S)-4), 139 mg (85.6%), mp
69-70 °C (ethyl acetate/hexane). 1H NMR (CDCl3), δ 1.50
The [18F]fluoride used for radiosyntheses was produced at
Emory University with a 11 MeV Siemens RDS 112 negative-
ion cyclotron (Knoxville, TN) by the 18O (p,n) 18F reaction
using [18O]H2O (95%). Trap/release cartridges model DW-TRC
were purchased from D&W, Inc. (Oakdale, TN). Alumina N
SepPak and HLB Oasis reverse phase cartridge were purchased
from Waters, Inc. (Milford, MA). Ion-retardation resin (AG
11A8 50-100 mesh) was purchased from BioRad (Hercules,
CA). The 0.2 μm Gelman Teflon filters were purchased from
VWR. Isolated radiochemical yields were determined using a
dose-calibrator (Capintec CRC-712M). Thin layer chromato-
grams of the radiolabeled compounds were analyzed with a
Raytest System (model: Rita Star, Germany) using the same
type of TLC plates from Whatman.
All animal experiments were carried out under humane
conditions and were approved by the Institutional Animal Use
and Care Committee (IUCAC) and Radiation Safety Commit-
tees at Emory University.
Chemistry. (S)- and (R)-2-N-(tert-Butoxycarbonyl)amino-2-
methyl-3-hydroxy-propanoic Acid (1). (S)-(þ)-2-Amino-2-meth-
yl-3-hydroxy-propanoic acid or (R)-(-)-2-amino-2-methyl-3-
hydroxy-propanoic acid [(S)- or (R)-R-methyl-serine] (773 mg,
6.48 mmol, Acros Organics, Morris Plains, NJ) was suspended
in 20 mL methanol-triethylamine-1N sodium hydroxide (9:1:1
v/v/v) and di-tert-butyl dicarbonate (2828 mg, 12.96 mmol) was
added in one portion. The reaction was stirred at room tem-
perature overnight. The organic solvent was removed under
reduced pressure, and 10 mL of ethyl acetate was added. The pH
of the aqueous phase was adjusted to 2 with 3N hydrochloric
acid. The organic layer was retained while the aqueous layer was
saturated with sodium chloride and extracted with ethyl acetate
(3 ꢀ 10 mL). The combined organic phases were dried over
magnesium sulfate, filtered, and concentrated to dryness under
reduced pressure. The product (S)- or (R)-1 was given as white
solid, which were used without further purification. (S)-2-
N-(tert-Butoxycarbonyl)amino-2-methyl-3-hydroxy-propanoic
acid ((S)-1), 1185 mg (83.5%), mp 114-117 °C (decomposed).
1H NMR (CDCl3) δ 1.47 (9H, s), 1.52 (3H, s), 3.79, 3.82 (1H, d,
J = 12 Hz), 3.93, 3.96 (1H, d, J = 12 Hz), 5.51 (1H, broad s).
(R)-2-N-(tert-butoxycarbonyl)amino-2-methyl-3-hydroxy-propanoic
acid ((R)-1), 1400 mg (quantitative), mp 115-117 °C
(decomposed). 1H NMR (CDCl3) δ 1.46 (9H, s), 1.51 (3H,
s), 3.81, 3.84 (1H, d, J = 12 Hz), 3.91, 3.94 (1H, d, J = 12 Hz),
5.50 (1H, broad s).

884 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Yu et al.
1
(9H, s), 1.57 (9H, s), 1.74 (3H, s), 4.26, 4.29 (1H, d, J = 9.6 Hz),
4.56, 4.58 (1H, d, J = 9.6 Hz). HRMS, m/z, calcd for C13H27-
N2O7S [M þ NH4]þ, 355.15335; found 355.15341 (100%).
(R)-3-(tert-Butoxycarbonyl)-4-methyl-1,2,3-oxathiazolidine-
4-carboxylic acid tert-butyl ester 2,2-dioxide ((R)-4), 138 mg
(85.3%), mp 70-71 °C (ethyl acetate/hexane). 1H NMR
(CDCl3), δ 1.50 (9H, s), 1.57 (9H, s), 1.74 (3H, s), 4.26, 4.29
(1H, d, J = 9.3 Hz), 4.56, 4.59 (1H, d, J = 9.3 Hz), Anal.
(C13H23NO7S) C, H, N.
(R)- and (S)-2-Amino-3-fluoro-2-methylpropanoic Acid (5),
Hydrochloride Salt. Compounds (R)- and (S)-5 were prepared
from (S)- and (R)-4, respectively, using the method previously
reported.22 Briefly, to a solution of the cyclic sulfamidate (S)- or
(R)-4 (30 mg, 0.089 mmol) in 1 mL of acetonitrile was added
tetrabutylammonium fluoride (71 mg, 0.27 mmol, 270 μL of
1.0 M solution in tetrahydrofuran), and the resulting solution
was stirred overnight at room temperature. The reaction mix-
ture was concentrated under reduced pressure, and the residue
was treated with 5 mL of 3N hydrochloric acid at 85 °C for 1 h.
After cooling, the aqueous solution was washed twice with 5 mL
of ether and then concentrated under reduced pressure to give
(R)- or (S)-5-HCl as white solid. (R)-2-Amino-3-fluoro-
2-methylpropanoic acid, hydrochloride salt ((R)-5-HCl), 7 mg
(49.9%), mp 204-206 °C (decomposed). 1H NMR (D2O) δ 1.50
(3H, s), 4.53-4.90 (2H, m). Anal. (C4H9ClFNO2) C, H, N. (S)-
2-Amino-3-fluoro-2-methylpropanoic acid, hydrochloride salt
((S)-5-HCl), 6.3 mg (44.9%), mp 202-205 °C (decomposed). 1H
NMR (D2O) δ 1.52 (3H, s), 4.54-4.92 (2H, m). Anal.
(C4H9ClFNO2) C, H, N.
(ethyl acetate /hexane). H NMR (CDCl3) δ 1.51 (9H, s), 1.53
(3H, s), 2.94 (3H, s), 4.21, 4.24 (1H, d, J = 8.7 Hz), 4.88, 4.91 (1H,
d, J = 8.7 Hz). Anal. (C9H17NO5S) C, H, N.
(R)- and (S)-3-Fluoro-2-methyl-2-(methylamino)propanoic
Acid (8), Hydrochloride Salt. (R)- and (S)-8 were prepared from
(S)- and (R)-7, respectively. To a solution of the cyclic sulfami-
date (S)- or (R)-7 (16 mg, 0.06 mmol) in 1 mL of acetonitrile was
added tetrabutylammonium fluoride (47 mg, 0.18 mmol, 180 μL
of 1.0 M solution in tetrahydrofuran), and the resulting solution
was stirred overnight at room temperature. The reaction mix-
ture was concentrated under reduced pressure, and the residue
was treated with 5 mL of 3N hydrochloric acid at 85 °C for 1 h.
After cooling, the aqueous solution was washed twice with 5 mL
of ether and then concentrated under reduced pressure to give
(R)- or (S)-8-HCl as white solid. (R)-3-fluoro-2-methyl-
2-(methylamino)propanoic acid, hydrochloride salt ((R)-8-
1
HCl), 5 mg (49%), mp 179-181 °C (decomposed). 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. (S)-3-Ffluoro-2-methyl-
2-(methylamino)propanoic acid, hydrochloride salt ((S)-8-
HCl), 4.1 mg (40%), mp 178-181 °C (decomposed). 1H NMR
(D2O) δ 1.47-1.49 (3H, m), 2.75 (3H, s), 4.65-4.92 (2H, m).
Anal. (C5H11ClFNO2) C, H, N.
Radiosynthesis of (R)- and (S)-2-Amino-3-[18F]fluoro-2-
methylpropanoic Acid ((R)- and (S)-[18F]5) and (R)- and (S)-
3-[18F]Fluoro-2-methyl-2-(methylamino)propanoic Acid ((R)-
and (S)-[18F]8). The same conditions were used to prepare
FAMP (R)-[18F]5 from (S)-4, (S)-[18F]5 from (R)-4, NMe-
FAMP (R)-[18F]8 from (S)-7, and (S)-[18F]8 from (R)-7, respec-
tively. The preparation of (R)- and (S)-[18F]5 as well as (R)- and
(S)-[18F]8 was based on the previously reported automated
synthesis of anti-[18F]FACBC, which involved a fully auto-
mated synthesis developed for the Siemens computer program-
mable chemistry process control unit (CPCU).31 The CPCU is a
valve-and-tubing system which is designed to accommodate one
or two glass reaction vessels and a number of reagent and
solvent reservoir containers. The automated production of the
18F-labeled amino acids was started by delivering 150-200 mCi
NCA [18F]HF from cyclotron target through a trap/release
cartridge T/R by using 0.9 mg (6.5 μmol) potassium carbonate
in 0.6 mL of water to a reaction vial containing 5 mg (13.3 μmol)
of Kryptofix 222 in 1 mL of acetonitrile. The solvent was
removed at 110 °C with nitrogen gas flow. Acetonitrile (1 mL)
was added, and the drying was repeated three times to remove
residual water. A 1 mg portion of the appropriate cyclic
sulfamidate 4 or 7 in 1 mL of acetonitrile was added to the vial,
and the reaction mixture was heated at 90 °C for 10 min. After
acidic hydrolysis using 0.5 mL of 4N hydrochloric acid at 110 °C
for 10 min, the product was purified47 by passing serially
through an AG 11A8 ion retardation resin column, an alumina
N SepPak, an HLB Oasis reverse phase cartridge, and a 0.2 μm
Gelman Teflon filter with sterile normal saline into a dose vial,
which was ready for use in vitro and in vivo studies. The identity
of the radiolabeled product was confirmed by comparing the
Rf value of the radioactive product visualized with radiometric
TLC with the Rf value of the authentic 19F compound visualized
with ninhydrin stain, with a solvent of acetonitrile/water/metha-
nol = 4:1:1 (v/v/v) (Rf = 0.4 for 5, Rf = 0.5 for 8). In all
radiosyntheses, the only peak present on radiometric TLC
analysis corresponded to 5 or 8, and the radiochemical purity
of the product exceeded 99%.
(S)- and (R)-4-Methyl-1,2,3-oxathiazolidine-4-carboxylic
Acid tert-Butyl Ester 2,2-Dioxide (6). To a solution of (S)- or
(R)-4 (161 mg, 0.478 mmol) in 2 mL of tert-butyl acetate-
dichloromethane (4:1 v/v) was added methanesulfonic acid
(138 mg, 1.43 mmol) at room temperature under an argon
atmosphere. After stirring at room temperature overnight,
5 mL of saturated sodium bicarbonate was added to the mixture.
The aqueous phase was extracted with ethyl acetate (3 ꢀ 10 mL).
The combined organic phases were washed with brine (1 ꢀ
10 mL), dried over sodium sulfate, and concentrated under
reduced pressure. The crude product was purified with silica
gel chromatography (20% ethyl acetate in hexane) to give 6 as
colorless oil. (S)-4-Methyl-1,2,3-oxathiazolidine-4-carboxylic
acid tert-butyl ester 2,2-dioxide ((S)-6), 64 mg (56.5%). 1H
NMR (CDCl3) δ 1.53 (9H, s), 1.63 (3H, s), 4.27, 4.30 (1H, d,
J = 9.6 Hz), 4.69, 4.72 (1H, d, J = 9.6 Hz), 5.51 (1H, broad s).
Anal. (C8H15NO5S) C, H, N. (R)-4-Methyl-1,2,3-oxathiazolidine-
4-carboxylic acid tert-butyl ester 2,2-dioxide ((R)-6), 56.1 mg
1
(49.5%). H NMR (CDCl3) δ 1.53 (9H, s), 1.63 (3H, s), 4.272,
4.30 (1H, d, J = 9.6 Hz), 4.69, 4.72 (1H, d, J = 9.6 Hz), 5.49 (1H,
broad s). Anal. (C8H15NO5S) Calcd C: 40.50, H: 6.37, N: 5.90.
Found C: 41.08, H: 6.41, N: 5.78.
(S)- and (R)-3,4-Dimethyl-1,2,3-oxathiazolidine-4-carboxylic
Acid tert-Butyl Ester 2,2-Dioxide (7). To a solution of (S)- or (R)-
6 (22 mg, 0.093 mmol) in 1 mL of tetrahydrofuran was added
sodium hydride (9 mg, 0.37 mmol) at room temperature under
an argon atmosphere and the mixture was stirred for 10 min
after which dimethyl sulfate (47 mg, 0.37 mmol) was added
dropwise. The mixture was stirred at room temperature over-
night. Water (2 mL) was added, and the mixture was extracted
with ethyl acetate (2 ꢀ 5 mL). The combined organic phases
were dried over sodium sulfate and concentrated under reduced
pressure. The crude product was purified by silica gel column
eluted with 20% ethyl acetate in hexane to give the title
compound as white solid. (S)-3,4-Dimethyl-1,2,3-oxathiazolidine-
4-carboxylic acid tert-butyl ester 2,2-dioxide ((S)-7), 13.3 mg
(56.9%), mp 84-85 °C (ethyl acetate/hexane). 1H NMR (CDCl3)
δ 1.51 (9H, s), 1.53 (3H, s), 2.94 (3H, s), 4.22, 4.24 (1H, d,
J = 8.4 Hz), 4.88, 4.90 (1H, d, J = 8.4 Hz). Anal. (C9H17NO5S)
C, H, N. (R)-3,4-Dimethyl-1,2,3-oxathiazolidine-4-carboxylic acid
tert-butyl ester 2,2-dioxide ((R)-7), 15.5 mg (66.4%), mp 84-85 °C
Amino Acid Uptake and Inhibition Assays. These assays were
performed by a modified reported method,22,38 and the proce-
dures are summarized here. The 9L rat gliosarcoma cell line was
cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
supplemented with 10% fetal calf serum, 100 units/mL penicillin,
and 100ug/mL streptomycin. Cells were maintained in
T-150 tissue culture flasks under humidified incubator condi-
tions (37 °C, 5% CO2/95% air) and were routinely passaged at
confluence. For the cell uptake experiment, 9L cells were

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 885
dispersed with a 0.05% solution of trypsin/ethylenediamine-
tetraacetic acid (EDTA), washed with Hank’s balanced salt
solution (HBSS) twice, and adjusted to a final concentration
of 5 ꢀ 107 cells/mL in HBSS.
point for each tracer using 1-way ANOVA with ProStat
software.
Acknowledgment. We are grateful to Dr. Bing Wang,
NMR Center of Emory University, for the help with NMR
instrumentation, sample treatment, and data analysis. We
acknowledge the use of Shared Instrumentation provided by
grants from the NIH and the NMP for the mass spectrometry
data.
In this study, approximately 5 ꢀ 105 cells were exposed to
5 μCi (R)-[18F]5, (S)-[18F]5, (R)- [18F]8, or (S)-[18F]8 in amino
acid free HBSS (0.1 mL) with or without transport inhibitors
(10 mM final concentration of BCH, MeAIB or ACS, re-
spectively) for 30 min under incubator conditions in 1.5 mL
conical tubes. Each assay condition was performed in triplicates.
After incubation, cells were twice centrifuged (75 G for 5 min)
and rinsed with ice-cold amino-acid/serum-free HBSS to re-
move residual activity in the supernatant. The activity in tubes
was counted in a Packard Cobra II Auto-Gamma counter, the
raw counts decay corrected, and the activity per cell number
determined. The data from these studies (expressed and norma-
lized as percent uptake relative to control per 5 ꢀ 105 cells) were
graphed using Excel, with statistical comparisons using 1-way
ANOVA with ProStat software (Poly Software International,
Pearl River, NY) between the groups analyzed.
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