Synthesis, radiolabeling, and biological evaluation of (R)- and (S)-2-amino-5-[18F]fluoro-2-methylpentanoic acid ((R)-, (S)-[18F]FAMPe) as potential positron emission tomography tracers for brain tumors

Article abstract of DOI:10.1021/jm502023y

A novel ¹⁸F-labeled α,α-disubstituted amino acid-based tracer, 2-amino-5-[¹⁸F]fluoro-2-methylpentanoic acid ([¹⁸F]FAMPe), has been developed for brain tumor imaging with a longer alkyl side chain than previously reported c

Full text of DOI:10.1021/jm502023y

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Article
Synthesis, radiolabeling, and biological evaluation of (R)- and (S)-2-
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amino-5-[F]fluoro-2-methylpentanoic acid ((R)-, (S)-[F]FAMPe) as
potential positron emission tomography tracers for brain tumors
Ahlem Bouhlel, Dong Zhou, Aixiao Li, Liya Yuan, Keith Rich, and Jon McConathy
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm502023y • Publication Date (Web): 06 Apr 2015
Downloaded from http://pubs.acs.org on April 12, 2015
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Synthesis, radiolabeling, and biological evaluation of (R)-
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and (S)-2-amino-5-[ F]fluoro-2-methylpentanoic acid ((R)-,
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(S)-[ F]FAMPe) as potential positron emission tomography
tracers for brain tumors
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Bouhlel, Ahlem ; Zhou, Dong ; Li, Aixiao ; Yuan, Liya ; Rich, Keith ; McConathy, Jonathan .
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Department of Radiology, Washington University in Saint Louis, School of Medicine, Missouri
Department of Neurosurgery, Washington University in Saint Louis, School of Medicine, Missouri.
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Abstract
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A novel Fꢀlabeled α,αꢀdisubstituted amino acidꢀbased tracer, 2ꢀaminoꢀ5ꢀ[ F]fluoroꢀ2ꢀ
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methylpentanoic acid ([ F]FAMPe) has been developed for brain tumor imaging with a longer alkyl side
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chain than previously reported compounds to increase brain availability via system L amino acid
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transport. Both enantiomers of [ F]FAMPe were obtained in good radiochemical yield (24ꢀ52% n= 8)
and high radiochemical purity (> 99%). In vitro uptake assays in mouse DBT gliomas cells revealed that
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(
S)ꢀ[ F]FAMPe enters cells partly via sodiumꢀindependent system L transporters and also via other nonꢀ
system A transport systems including transporters that recognize glutamine. Biodistribution and small
animal PET/CT studies in the mouse DBT model of glioblastoma showed that both (R)- and (S)-
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[
F]FAMPe have good tumor imaging properties with the (S)ꢀenantiomer providing higher tumor uptake
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and tumor to brain ratios. Comparison of the SUVs showed that (S)-[ F]FAMPe had higher tumor to
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brain ratios compared to (S)-[ F]FET, a wellꢀestablished system L substrate.
Introduction
Brain tumor imaging with the most widely used positron emission tomography (PET) tracer for
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oncology, the glucose analogue 2ꢀdeoxyꢀ2ꢀ[ F]fluoroꢀDꢀglucose ([ F]FDG), is often difficult to interpret
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due to the high physiologic uptake of [ F]FDG in normal brain and variable [ F]FDG uptake in regions
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ꢀ3
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showing postꢀtreatment effects such as radiation necrosis. In contrast to [ F]FDG, radiolabeled amino
acid tracers targeting system L amino acid transporters have superior brain tumor imaging properties due
1, 4
to upregulation of amino acid transporters in gliomas and lower uptake in normal brain. These tracers
complement anatomic imaging with magnetic resonance imaging (MRI) for definition of tumor margins,
monitoring response to therapy, and distinguishing treatment effects from recurrent tumor. System L
preferentially transports amino acids with large, neutral side chains (e.g. Lꢀphenylalanine, Lꢀtyrosine, Lꢀ
leucine) and has four family members: LAT1, LAT2, LAT3, and LAT4. LAT1 and LAT2 are sodiumꢀ
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independent and function by exchanging one intracellular amino acid for one extracellular amino acid
whereas LAT3 and LAT4 mediate facilitated diffusion of substrates across membranes. System L
transporters are active at the luminal side of the bloodꢀbrain barrier (BBB) which allows system L
substrates to reach the entire tumor volume even when the BBB is not disrupted. A number of wellꢀ
established PET tracers targeting system L transport have been used for human brain tumor imaging
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including Lꢀ[ C]methionine ([ F]MET), Oꢀ(2ꢀ[ F]fluoroethyl)ꢀLꢀtyrosine ([ F]FET) and 6ꢀ[ F]fluoroꢀ
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4ꢀ12
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,4ꢀdihydroxyꢀLꢀphenylalanine ([ F]FDOPA).
Certain radiolabeled amino acids have also shown
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promising imaging properties for other types of tumors such as neuroendocrine tumors with [ F]FDOPA
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13ꢀ
and prostate cancer with antiꢀ1ꢀaminoꢀ3ꢀ[ F]fluorocyclobutylꢀ1ꢀcarboxylic acid (antiꢀ3ꢀ[ F]FACBC).
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A range of amino acid transporter systems and substrates have been shown to be relevant to
oncologic imaging, and there has been recent interest in PET tracers targeting glutamine transport and
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metabolism. The metabolically inert tracer anti-3-[ F]FACBC has been shown to be a substrate for
ASCT2, an important glutamine transporter in cancer, and to a lesser extent system L and system A, in
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human prostate cancer cells.
Human studies indicate that anti-3-[ F]FACBC has clinical potential for
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imaging prostate cancer and brain tumors.
Another strategy for targeting glutamine metabolism in
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cancer has employed a series of F-labeled analogues of glutamine. In particular, 4-[ F]-(2S,4R)-
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fluoroglutamine ([ F]-FGln) has been shown to be promising in human studies for monitoring
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progression of gliomas. Additionally, the F-tracer targeting system ASC, 3-(1-[ F]fluoromethyl)-L-
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alanine (L-[ F]FMA) was shown to be a potential useful PET tracer in preclinical studies. A limitation of
the tracers FGln and FMA has been in vivo defluorination. There has also been recent interest in
glutamate analogues and other amino acids bearing carboxylic acids on their side chains targeting
-
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system x which is upregulated in many cancers and a marker of tumor oxidative stress.
Finally, lead
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compounds targeting cationic amino acid transporters including (S)-2-amino-3-[1-(2-[ F]fluoroethyl)-1H-
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[
1,2,3]triazol-4-yl]propanoic acid ((S)-[ F]AFETP) and N-2-[ F]fluoroethyl-N-methyl-2-aminoethyl-
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tyrosine ([ F]FEMAET) have been recently developed and reported.
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An important limitation of system L transport substrates as imaging agents is the inability of
system L transporters to directly concentrate substrates intracellularly. This property leads to relative low
tumor to tissue ratios which can reduce sensitivity for lesion detection and lead to washout of activity
from tumors over time. Other amino acid transporters including system A transporters can concentrate
substrates intracellularly, providing higher and more persistent uptake in tumors. However, the lack of
activity of many amino acid transporters at the luminal side of the BBB limits access of their substrates to
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the enhancing regions of brain tumors. These observations prompted us to develop Fꢀlabeled amino acid
tracers that target both system L and nonꢀsystem L amino acid transporters in an effort to maintain brain
availability through system L transport while enhancing tumor to normal tissue ratios through nonꢀsystem
L transporters. In our previously reports of the synthesis and biological evaluation of three α,αꢀ
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disubstituted amino acids with varying selectivities for system A and system L transporters: 3ꢀ[ F]fluoroꢀ
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2ꢀmethylꢀ2ꢀNꢀ(methylamino)propanoic acid ([ F]MeFAMP), 2ꢀaminoꢀ3ꢀ[ F]fluoroꢀ2ꢀmethylpropanoic
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acid ([ F]FAMP), and 2ꢀaminoꢀ4ꢀ[ F]fluoroꢀ2ꢀmethylbutanoic acid ([ F]FAMB),
we proposed to
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develop new Fꢀlabeled analogues with longer alkyl chain lengths to increase recognition by system L
transporters and thereby improve brain availability.
Here, we report efficient organic and radiosynthetic routes to obtain the (R)- and the (S)-
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enantiomers of 2ꢀaminoꢀ5ꢀ[ F]fluoroꢀ2ꢀmethylpentanoic acid ([ F]FAMPe), an analogue of [ F]FAMP
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and [ F]FAMB. These novel tracers were evaluated in the mouse DBT (delayed brain tumor) model of
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glioblastoma through in vitro uptake assays, biodistribution studies, and small animal PET/CT imaging.
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The tumor and the brain uptake of (R)- and (S)-[ F]8 ((R)ꢀ and (S)ꢀ[ F]FAMPe) were compared with
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the established system L PET tracer, (S)-[ F]FET, and the system A PET tracer (R)-[ F]MeFAMP
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through small animal PET studies. (S)-[ F]8 demonstrated very high uptake in this tumor model and
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increased recognition by system L transport compared to (R)-[ F]MeFAMP. However, the longer side
chain led to loss of recognition by system A transporters with recognition by other neutral amino acid
transporters including glutamine transport systems.
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A.
B.
O
O
Me
HN
Me
O
O
O
H N
H2N
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H N
H N
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OH
OH
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OH
OH
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Me
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[18F]FAMP
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F
[18
F]MeFAMP
[18F]FAMB
HO
O
OH
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C.
O
O
(
S)-[ F]FET
(S)-[ F]FDOPA
H N
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H N
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OH
OH
Me
Me
18F
18F
(R)-[ F]FAMPe
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(
S)-[ F]FAMPe
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Figure 1: Selected Fꢀlabeled amino acids. (A) Both [ F]FET and [ F]FDPOA have been used for imaging brain
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tumors in human subjects. (B) [ F]FAMP, [ F]FAMB, and [ F]MeFAMP are α,αꢀdisubstituted amino acids; (R)ꢀ
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[
F]MeFAMP is an established substrate for system A transport. (C) (S)ꢀ and (R)ꢀ[ F]FAMPe are novel α,αꢀ
disubstituted amino acids evaluated in this publication in the DBT glioma model.
Results and Discussion
1
. Chemistry.
The racemic nonꢀradioactive forms of the amino acid 2ꢀaminoꢀ5ꢀfluoroꢀ2ꢀmethylpentanoic acid
(R,S)ꢀ8 ) were prepared in a simple eight step synthesis as shown in Scheme 1. From commercially
(
available Lꢀalanine tertꢀbutyl ester hydrochloride and benzophenone imine, we synthesized in very good
yield (94%) and without further purification our first intermediate which is the ester imine derivative of
alanine, tertꢀbutyl 2ꢀ((diphenylmethylene)amino)propanoate ((S)-1). In a second step, we conducted a
nonꢀstereoselective alkylation with allyl iodide at the αꢀcarbon of the amino acid derivative which
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provided tertꢀbutyl (R,S)ꢀ2ꢀ((diphenylmethylene)amino)ꢀ2ꢀmethylpentꢀ4ꢀenoate ((R,S)-2), also in very
good yield (90%). After the simple and rapid hydrolysis of the imine function of (R,S)-2 with
hydroxylamine hydrochloride at room temperature, we obtained the amino acid ester tertꢀbutyl (R,S)ꢀ2ꢀ
aminoꢀ2ꢀmethylpentꢀ4ꢀenoate ((R,S)-3). The following step protected this amine in presence of diꢀtertꢀ
butyl dicarbonate which gave tertꢀbutyl (R,S)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ2ꢀmethylpentꢀ4ꢀenoate
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(
(R,S)-4) in very good yield (92%). Then, an hydroboration of the alkene followed by hydrolysis in
presence of 30% hydrogen peroxide provided the desired terminal alcohol intermediate, tertꢀbutyl (R,S)ꢀ
ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ5ꢀhydroxyꢀ2ꢀmethylpentanoate ((R,S)-5) in a good yield (62%). The
2
alcohol was converted into a leaving group through tosylation to provide the racemic labeling precursor
tertꢀbutyl (R,S)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ2ꢀmethylꢀ5ꢀ(tosyloxy)pentanoate ((R,S)-6) in moderate
yield (40%).
To perform the nucleophilic substitution of the tosylate group with nonꢀradioactive fluoride, we
tried several different conditions. Our first attempt consisted of treating the tosylate compound (R,S)-6
with an excess of tetrabutylammonium fluoride in acetonitrile at room temperature. The reaction
consumed the starting material to provide a single major product in 86%, but instead of the desired
product, an intramolecular cyclization occurred to form diꢀtertꢀbutyl (R,S)ꢀ2ꢀmethylpyrrolidineꢀ1,2ꢀ
dicarboxylate ((R,S)-9) as shown in Scheme 2. This reaction gave only trace quantities of the desired
fluoro product tert-butyl (R,S)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ5ꢀfluoroꢀ2ꢀmethylpentanoate ((R,S)-7).
Because a similar reaction proceeded without this difficulty in the synthesis of FAMB (one carbon shorter
32
side chain), the favorable kinetics of the intramolecular formation of a five ring cycle is the likely cause
in this case. Attempts to form the diꢀBoc protected nitrogen compound as has been reported in the
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5
synthesis of fluoroleucine derivatives were not successful. We thus followed a different protocol in
which the authors used tertꢀamyl alcohol (2ꢀmethylbutanꢀ2ꢀol) as solvent for nucleophilic fluorination
36
reactions. The authors suggested that the tertꢀamyl alcohol solvent system might have four beneficial
influences on the mechanism of this fluorination process: (i) the hydrogen bond between the tertꢀamyl
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alcohol solvent and the fluoride may reduce the strength of the CsF ionic bond, (ii) the solvation of
fluoride will be limited and thus fluoride will be a good nucleophile due to the bulky tertꢀamyl alcohol,
(
iii) the hydrogen bond between the tertꢀamyl alcohol solvent and the oxygen atoms of a sulfonate leaving
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group may enhance the leaving group reactivity, and (iv) the protic environment of the reaction medium,
as well as hydrogen bonding between the tertꢀamyl alcohol and reactive heteroatoms in the substrate, may
reduce side reactions such as intramolecular cyclization. By using three equivalents of cesium fluoride
(
CsF) in tertꢀamyl alcohol (2ꢀmethylbutanꢀ2ꢀol) at 80 °C, we obtained the desired fluoro compound (R,S)-
and also the cyclic compound (R,S)-9 after 1 h, but approximately 15% of starting material (R,S)-6
7
remained. By increasing the amount of CsF used to five equivalents and the temperature to 100 °C
without reaching reflux, the reaction was completed in 1 h, and the fluoro compound (R,S)-7 was obtained
in 34% yield. We also conducted the reaction in the presence of ten equivalents of CsF but the same yield
was obtained. Thus, Kim’s methodology allowed us to obtain the desired product (R,S)-7 in acceptable
yields. The final step consisted of the deprotection of (R,S)-7. The deprotection was achieved with 4 M
hydrochloric acid at 60 °C which provided the racemic nonꢀradioactive amino acid (R,S)-8 as the
hydrochloride salt in good yield (78%) and high purity.
Scheme 1: Multistep synthesis of the racemic nonꢀradioactive amino acid (R,S)ꢀ8.
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Reagents and conditions: a) diphenylmethanimine, DCM, rt, 2 h; b) LDA, 3ꢀiodopropꢀ1ꢀene, THF, ꢀ78 °C then rt for 18 h; c)
NH OH, MeOH, rt, 1 h; d) Boc O, THF, rt, 18 h; e) BH .THF then NaOH 1 M, H O 30%, 0 °C then rt for 18 h; f) TsCl,
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3
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2
NaOtBu, DCM, 0 °C then rt for 18 h; g) CsF, 2ꢀmethylbutanꢀ2ꢀol, 100 °C, 1h; h) HCl 4 M, 60 °C, 90 min.
Scheme 2: Reaction conditions (equivalents of fluoride, solvent and temperature) were optimized to minimize the
cyclized compound (R,S)-9.
To obtain the enantiomerically pure radiolabeling precursors, tosylate precursors (S)-6 and (R)-6 were
prepared in four steps started with the commercially available starting materials (S)ꢀ(ꢀ)ꢀαꢀallylalanine and
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(
R)ꢀ(+)ꢀαꢀallylalanine respectively (see Scheme 3). During the first step, (S)ꢀαꢀallylalanine and (R)ꢀαꢀ
allylalanine were treated with diꢀtertꢀbutyl dicarbonate to give the carbamates (R)- and (S)-2ꢀ((tertꢀ
butoxycarbonyl)amino)ꢀ2ꢀmethylpentꢀ4ꢀenoic acid ((S)-10 and (R)-10), respectively, which were used
without purification in the next reaction. The second step consisted of the protection of the carboxylic
acid function of (S)-10 and (R)-10 by using N,Nꢀdimethylformamide diꢀtertꢀbutyl acetal; compounds (S)-4
and (R)-4 were thus obtained. Then, as previously described for the racemic mixture (R,S)-4, the alkene
on the alkyl chain was converted into a terminal alcohol which provided products (S)-5 and (R)-5. In the
last step, these compounds were converted into tosylates to provide the radiolabeling precursors (S)-6 and
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(
R)-6. Each enantiomerically pure radiolabeling precursor was obtained in similarly good yield.
Scheme 3: Synthesis of the tosylate precursors (S)-6 and (R)-6 for radiolabeling.
Reagents and conditions: a) Boc O, MeOH/NEt /NaOH 1 M for 18 h; b) N,Nꢀdimethylformamide diꢀtert-butyl acetal, anhyd
2
3
toluene for 2 h at 90 °C then 18 h at rt; c) BH .THF then NaOH 1 M, H O 30%, 0 °C then rt for 18 h; f) TsCl, NaOtBu, DCM, 0
3
2
2
°C then rt for 18 h.
2. Radiochemistry.
1
8
The radiosynthesis was conducted in two steps (see Scheme 4). The first step, nucleophilic [ F]fluoride
substitution, was successfully performed in tertꢀamyl alcohol (2ꢀmethylbutanꢀ2ꢀol) at 96ꢀ105 °C to afford
18
18
the intermediates (S)-[ F]7 and (R)-[ F]7 from the appropriate enantiomerically pure precursor. Using
1
8
solid phase extraction with a C cartridge, the Fꢀlabeled intermediate was retained while the unreacted
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[
F]fluoride was removed. Then, the Fꢀlabeled intermediate was eluted from the cartridge with
acetonitrile and purified by reverseꢀphase high performance liquid chromatography (HPLC). The
1
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collected fractions from HPLC containing the Fꢀlabeled intermediate were combined, diluted with
0
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water, and passed through a light hydrophilicꢀlipophilicꢀbalanced (HLB) cartridge which was rinsed two
1
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more times with water in order to remove residual acetonitrile. The intermediates, (S)-[ F]7 or (R)-[ F]7,
were then eluted with a small amount of ethanol. In a second step, a quantitative deprotection was
achieved with aqueous hydrochloric acid and followed by elution through a cartridge allowing the
1
8
18
neutralization of highly acidic sample to provide the final product (S)-[ F]8 or (R)-[ F]8 which were
obtained in 24% to 52% (n= 8) decay corrected yields respectively, at pH= 6 and over 99% radiochemical
purity determined by radiometric thinꢀlayer chromatography and analytic HPLC (see Figure 2). In a
1
8
representative synthesis, a total of 9.9 mCi of (R)-[ F]8 at end of synthesis (EOS) was obtained from 40.8
1
8
mCi of [ F]fluoride after the drying process in a synthesis time of approximately 120 min using 5 mg of
the tosylate precursor (R)-6. The specific activity was > 1 Ci/µmol at the end of synthesis. Sterile water
was used for formulation of the final product for cell uptake assays to avoid the presence of sodium ions
in the sodiumꢀfree uptake conditions. For the animal studies, the solution was passed through a 0.22 µm
1
8
nylon filter, and then adjusted to a concentration of 0.9% sodium chloride. The stability of (S)-[ F]8 and
18
(R)-[ F]8 up to 6 h after formulation was confirmed by analytical HPLC.
18
Scheme 4: Radiosynthesis of (S)ꢀ[ F]8.
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Figure 2: Analytical chiral HPLC coꢀinjections of a mixture of the nonꢀradioactive racemic product (R,S)-8 (seen
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with UV detection) and (S)-[ F]8 (seen with radiometric detection).
3
. Cell uptake assays.
Amino acid uptake assays were performed using mouse DBT glioma cells in the presence and
absence of two wellꢀdescribed inhibitors of amino acid transport, Nꢀmethyl αꢀaminoisobutyric acid
MeAIB) and 2ꢀaminoꢀbicyclo[2.2.1]ꢀheptaneꢀ2ꢀcarboxylic acid (BCH), which were used as inhibitors for
system A and system L, respectively. These two commonly used inhibitors allowed us to evaluate the
(
1
8
contribution of these amino acid transport systems to the cellular uptake of (S)- and (R)-[ F]8. The results
of the in vitro cell uptake assays are shown in Figure 3. The two enantiomers were compared to (R)-
18
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[
F]MeFAMP, a good substrate for system A amino transport, and (S)-[ F]FET, a wellꢀcharacterized
system L transport substrate. The uptake assays were performed with a brief (30 or 60 seconds) uptake
18
time to evaluate the initial influx of (S)- or (R)-[ F]8 and minimize the potential for efflux which occurs
with many amino acid transporters including system L and ASC. An additional set of inhibition
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experiments shown in Figure 4 were performed using varying concentrations of Lꢀglutamine (Gln) to
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establish the role of glutamine transporters in the uptake of (S)- and (R)-[ F]8.
Certain amino acids transporters including system A and system ASC require coꢀtransport of
sodium ions to function while others including system L function in sodiumꢀfree conditions. Thus, we
compared uptake in the presence of sodium ions and under sodiumꢀfree conditions using the cation
choline as a substitute for sodium ions. In presence of MeAIB, there was no significant inhibition uptake
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of (S)-[ F]8, indicating that system A transport does not play a substantial role in the in vitro uptake of
1
8
this tracers by DBT cells. A minor component of inhibition of (R)-[ F]8 was observed with MeAIB (83 ±
17% uptake relative to control, p < 0.05). These results are similar to the ones obtained with the
18
structurally similar compound, [ F]FMA, developed by Wang et al., as uptake of this tracer by 9L
2
5
glioma cells was not inhibited by MeAIB. Substitution of choline for sodium ions resulted in partial
1
8
inhibition of uptake of both enantiomers with 71 ± 17 % uptake relative to control for (S)-[ F]8 and 70 ±
18
1
1% for (R)-[ F]8. These results indicate that both enantiomers enter DBT cells partly via sodiumꢀ
dependent amino acid transport, but not via the sodiumꢀdependent system A, as shown by the lack of
uptake inhibition by MeAIB. In the presence of MeAIB, the uptake of the system A substrate (R)-
18
[ F]MeFAMP was 19 ± 9% relative to the sodium control which is consistent with prior reports that (R)-
18
31, 33, 37
[ F]MeFAMP is a good substrate for system A in a range of rodent and human cancer cell lines.
1
8
Similarly, the uptake of (R)-[ F]MeFAMP was reduced to 21 ± 19% relative to the sodium control in the
sodiumꢀfree choline condition, consistent with sodiumꢀdependence of system A transport and
1
8
demonstrating that (R)-[ F]MeFAMP enter DBT cells mainly via the system A amino acid transport. As
1
8
expected, uptake of the system L transport substrate (S)-[ F]FET by DBT cells was not significantly
inhibited by MeAIB, inhibitor of the system A amino acid transporter, or by sodiumꢀfree conditions as
system L is sodiumꢀindependent.
BCH is commonly used as a competitive antagonist of the sodiumꢀindependent L type transport
0,+
0
system, but also competitively inhibits amino acid uptake via the sodiumꢀdependent B and B transport
1
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system. In the presence of sodium, BCH reduced the uptake of (S)-[ F]8 to 59 ± 15% relative to control
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and reduced the uptake of (R)-[ F]8 to 47 ± 12% relative to control. In the absence of sodium, the
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addition of BCH reduced the uptake of (S)-[ F]8 and (R)-[ F]8 to 72 ± 11% (p = 0.017) and 46 ± 4% (p
<
0.001) respectively, relative to the choline control condition. These results indicate a component of
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system L transport of both compounds. In the case of (R)-[ F]MeFAMP, no significant inhibition was
observed in the presence of sodium BCH relative to control. In contrast, almost all of the uptake of (S)-
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[
F]FET was inhibited by BCH in the presence and absence of sodium (p < 0.01), consistent with prior
18
9
studies showing (S)-[ F]FET is substrate for system L amino acid transport.
Equal concentrations of the amino acids alanine, serine, and cysteine (ASC) were also used for
uptake inhibition as these amino acids are competitive inhibitors for many neutral amino acid transporters
including system A, system ASC, and to a lesser extent system L. In the sodium ASC condition, the
1
8
18
uptake of (S)-[ F]8 and (R)-[ F]8 were reduced to 52 ± 14% and 41 ± 4%, respectively, relative to the
18
sodium control (p < 0.001 for both). These results indicate that (R)- and (S)-[ F]8 undergo uptake by
nonꢀsystem A neutral amino acid transport in addition to a component of system L transport. In the case
18
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of (R)-[ F]MeFAMP, the ASC condition reduced the uptake of (R)-[ F]MeFAMP to 22 ± 9% relative to
1
8
the sodium control (p < 0.001); the uptake of (S)-[ F]FET was reduced by approximately 40% but did not
reach statistical significance.
Because glutamine is an important amino acid in cancer metabolism and the ASC condition is
expected to inhibit glutamine uptake, additional uptake assays were performed in presence of different
concentrations of glutamine (0.1, 1.0 and 10 mM) and ASC (1 mM and 10 mM) as shown in Figure 4.
18
The results demonstrated that (S)-[ F]8 uptake was more sensitive to glutamine and ASC inhibition than
18
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(
R)-[ F]8. The uptake of (S)ꢀ[ F]8 was blocked in a doseꢀdependent manner by glutamine and ASC; in
1
8
contrast, significant inhibition of (R)-[ F]8 did not occur in the presence of glutamine and was observed
only with the 10 mM ASC condition. At the 1 mM and 10 mM concentration of glutamine, the uptake of
18
(
S)ꢀ[ F]8 was respectively reduced to 76 ± 21% (p < 0.05) and 66 ± 9% (p < 0.001) relative to the control
18
value. Similarly, at the 1 mM and 10 mM concentrations of ASC, the uptake of (S)ꢀ[ F]8 uptake was
respectively reduced to 61 ± 16% (p < 0.01) and 38 ± 9% relative to the control value. In contrast, only
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the 10 mM concentration of ASC caused a significant reduction of (R)ꢀ[ F]8 uptake (49 ± 9% relative to
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control, p < 0.001). These data suggest that (S)-[ F]8 uptake by DBT cells is partly mediated by amino
acid transporters that recognize glutamine.
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Figure 3: In vitro uptake of (S)- and (R)-[ F]8, (R)-[ F]MeFAMP and (S)-[ F]FET in DBT glioma cells in
presence and absence of competitive inhibitors of amino acid transport. The uptake data are normalized based on the
amount of activity added to each well and the total amount of protein in each well. The data are expressed as
percentage uptake relative to the sodium control condition, and the values for each condition are noted in the
18
appropriate bars. The number of replicates per condition were 7 or 8 for (S)ꢀ and (R)ꢀ[ F]8 and 3 or 4 for (R)ꢀ
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[
F]MeFAMP and (S)ꢀ[ F]FET. Na control and Cho control contain 10 mM sucrose to provide an osmolarity
consistent with the inhibitory conditions. Na= assay buffer containing sodium ions; Cho= assay buffer containing
choline ions; MeAIB= 10 mM Nꢀmethyl αꢀaminoisobutyric acid (system A inhibitor); BCH= 10 mM 2ꢀaminoꢀ
bicyclo[2.2.1]ꢀheptaneꢀ2ꢀcarboxylic acid (system L inhibitor); ASC= 3.3 mM each of LꢀAla, LꢀSer, LꢀCys. pꢀvalues
represent comparisons of uptake in the presence of inhibitor to control uptake for each radiotracer (1ꢀway ANOVA)
with Dunnett’s multiple comparison postꢀtests . The choline sucrose control condition was compared to the choline
BCH condition using a 2ꢀsided tꢀtests. * = p < 0.05, ** = p = 0.017, † = p <0.001, and †† = p < 0.01.
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1
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Figure 4: In vitro uptake of the two enantiomers (S)- and (R)-[ F]8 in DBT glioma cells in presence and absence of
competitive inhibitors of amino acid transport. The uptake data are normalized based on the amount of activity
added to each well and the total amount of protein in each well. The data are expressed as percentage uptake relative
to the sodium control condition, and the values for each condition are noted in the appropriate bars. Replicates per
condition (n= 8). To provide a consistent osmolarity compared to the inhibitory conditions, Na control contain 10
mM sucrose. Na= assay buffer containing sodium ions; Gln= glutamine; 10 mM ASC= 3.3 mM each of LꢀAla, Lꢀ
Ser, LꢀCys. pꢀValues represent comparisons of uptake in the presence of inhibitor to control uptake for each
radiotracer (1ꢀway ANOVA) with Dunnett’s multiple comparison postꢀtests. * = p < 0.05, ** = p < 0.001.
1
8
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Taken together, these in vitro inhibition studies indicate that (S)-[ F]8 and (R)-[ F]8 entered
DBT cells predominantly via sodiumꢀindependent amino acid transporters including a component of
18
system L transport. Additionally, (S)-[ F]8 uptake appears to be mediated in part by amino acid
transporters that recognize glutamine, although defining the specific transporter systems involved will
18
require further characterization. Because of the importance of glutamine in cancer metabolism, (S)-[ F]8
may serve as a lead compound for developing more selective probes for glutamine transport. There is a
growing evidence that certain system L and system ASC amino acid transporters, in particular LAT1 and
ASCT2, are involved in signaling including the mTOR pathways, promote tumor growth, and have
1, 4, 38ꢀ43
prognostic significance.
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4
. Biodistribution studies in mice with subcutaneous DBT glioma
18
The results of biodistribution studies with each enantiomer of [ F]8 in BALB/c mice with
subcutaneous DBT tumors 5, 30, and 60 min postꢀinjection (p.i.) are shown in Table 1 and expressed as
0
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percent of total injected dose per gram of tissue (%ID/g). The results demonstrated good tumor uptake of
1
8
radioactivity at the different time points p.i. for both (S)- and (R)-[ F]8 with progressive increase over
time for the (S)-enantiomer. The uptake in the brain also increases for both enantiomers but to a lesser
18
extent than the tumor. At each time point, the uptake of both (S)- and (R)-[ F]8 in subcutaneous glioma
was higher than normal brain (p < 0.001).
1
8
The tumor uptake of activity of (S)-[ F]8 after injection was very rapid with 5.1 ± 0.45 %ID/g at
1
8
5 min, and increased to 7.4 ± 0.87 %ID/g at 30 min and 9.9 ± 2.5 %ID/g at 60 min. For (R)-[ F]8, similar
1
8
tumor uptake was observed at 5 min with a value of 3.7 ± 0.60 %ID/g. The uptake of (R)-[ F]8 did not
1
8
18
increase to the same extent over time as with (S)-[ F]8, and (R)-[ F]8 led to 4.2 ± 0.49 %ID/g at 30 min
18
and 3.3 ± 0.65 %ID/g at 60 min. The tumor uptake of (R)ꢀ[ F]8 decreased slightly between 30 and 60
18
18
min while uptake of (S)ꢀ[ F]8 increased between these time points. The tumor uptake of (S)-[ F]8 was
18
significantly higher than with (R)-[ F]8 at 30 and 60 min after injection (p < 0.01 and p < 0.001,
1
8
respectively). Moreover, 1 h after injection the value of the uptake of (S)-[ F]8 was three time that of (R)-
1
8
18
18
[ F]8. Similarly, the normal brain uptake was significantly higher with (S)-[ F]8 than with (R)-[ F]8
with 0.65 ± 0.06 %ID/g 5 min after injection versus 0.48 ± 0.07 %ID/g, respectively (p < 0.05). The
uptake increased very little at 30 min post injection for both enantiomers, but increased more at 60 min p.i
1
8
18
with 0.85 ± 0.08 %ID/g for (S)-[ F]8 compared to 0.56 ± 0.13 %ID/g for (R)-[ F]8 (p < 0.001). The
1
8
18
biodistribution studies showed a higher activity in the blood for (S)-[ F]8 than for (R)-[ F]8 with 8.9 ±
1
8
0.3 %ID/g and 5.7 ± 0.7 %ID/g respectively at 5 min p.i. with slower clearance of (S)-[ F]8 than for (R)-
1
8
[ F]8 at the 60 min time point.
1
8
As shown in Figure 5, tumor to brain ratios for (S)-[ F]8 ranged from 7.9 to 11.8 and increased
18
over the course of the study. Tumor to brain ratios for (R)-[ F]8 started at 7.7, increased to 8.6 at 30 min
1
8
p.i., and finally decreased to 5.8 at 1 h. The tumor to brain ratios were higher for (S)-[ F]8 than for the Rꢀ
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enantiomer, primarily due to the higher tumor uptake of (S)-[ F]8. Differential transport and biological
properties of enantiomers of radiolabeled amino acids has been described in the literature for many
1
8
radiolabeled compounds. For example, the system A substrate (R)-[ F]MeFAMP showed higher tumor
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uptake than (S)-[ F]MeFAMP in a range of human tumor xenograft models. Our results demonstrate
the importance of assessment of the in vitro and in vivo biological properties for both enantiomers, as (S)-
18
[
F]8 appeared to be the more promising enantiomer for imaging due to substantially higher uptake in
tumors and normal brain.
18
Table 1: Biodistribution of (S)- and (R)-[ F]8 in BALB/c mice with subcutaneous DBT tumors.
5min
30min
60min
(S )-[F-18]FAMPe (R )-[F-18]FAMPe (S )-[F-18]FAMPe (R )-[F-18]FAMPe (S )-[F-18]FAMPe (R )-[F-18]FAMPe
blood
bone
brain
fat
8.86 +/- 0.31
2.69 +/- 0.17
0.65 +/- 0.06
1.42 +/- 0.19
3.24 +/- 0.49
14.89 +/- 2.89
4.30 +/- 0.38
9.10 +/- 0.63
2.10 +/- 0.13
5.67 +/- 0.75
2.25 +/- 0.73
0.48 +/- 0.07
1.77 +/- 0.62
2.21 +/- 0.43
18.90 +/- 2.21
2.74 +/- 0.29
7.37 +/- 0.83
1.52 +/- 0.28
25.68 +/- 4.21
4.28 +/- 0.76
3.13 +/- 0.81
3.68 +/- 0.60
4.82 +/- 0.38
3.96 +/- 0.29
0.68 +/- 0.06 ††
0.91 +/- 0.15
2.91 +/- 0.22
15.40 +/- 1.31
4.67 +/- 0.43
4.91 +/- 0.53
1.95 +/- 0.19
53.06 +/- 14.34
5.82 +/- 0.77
3.56 +/- 0.45
1.79 +/- 0.24
2.55 +/- 0.90
0.49 +/- 0.05 ††
1.15 +/- 0.54
1.50 +/- 0.36
8.04 +/- 1.60
2.31 +/- 0.26
2.85 +/- 0.40
1.15 +/- 0.29
21.16 +/- 6.22
2.51 +/- 0.41
1.74 +/- 0.30
3.88 +/- 0.42
6.33 +/- 0.96
0.85 +/- 0.08 *†
0.77 +/- 0.15
2.89 +/- 0.29
13.02 +/- 4.26
4.30 +/- 0.41
4.05 +/- 0.45
2.14 +/- 0.24
47.33 +/- 15.72
5.05 +/- 0.49
3.86 +/- 0.18
9.88 +/- 2.48 **
1.05 +/- 0.22
2.91 +/- 0.86
0.56 +/- 0.13 *†
0.41 +/- 0.13
1.16 +/- 0.19
4.72 +/- 1.06
1.46 +/- 0.39
1.65 +/- 0.40
1.00 +/- 0.17
11.62 +/- 3.90
1.96 +/- 0.42
1.39 +/- 0.40
3.27 +/- 0.65 **
†
†
heart
kidney
liver
lung
muscle
pancreas 34.09 +/- 5.07
spleen
thyroid
tumor
5.77 +/- 0.37
3.82 +/- 0.89
5.09 +/- 0.45
7.37 +/- 0.87
*
4.20 +/- 0.49
*
18
Mice were not anesthetized for the biodistribution studies. 23 µCi (0.85 MBq) of (S)ꢀ[ F]8, and 29 µCi (1.07 MBq)
18
of (R)ꢀ[ F]8 were administrated via tail vein injection. The animals were euthanized after 5, 30, and 60 min in
group of 5 animals. The data are expressed as %ID/g and errors are expressed as standard deviation. pꢀValues
1
8
represent comparisons of uptake data for (S)ꢀ versus (R)ꢀ[ F]8 in tumor and brain tissue through 1ꢀway ANOVA
with Tukey postꢀtests. * = p < 0.01, **, ††,*† = p < 0.001, † = p < 0.05.
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Figure 5: Tumor to brain ratios from biodistribution studies conducted in BALB/c mice with subcutaneous DBT
tumors.
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As for the brain and DBT tumor, (S)-[ F]8 demonstrated higher uptake than (R)-[ F]8 in most of
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the normal organs at all time points evaluated. The highest levels of uptake for (S)- and (R)-[ F]8 at all
time points were observed in the kidneys and pancreas, a pattern frequently seen with radiolabeled amino
acids. Although excretion in the urine and feces was not measured, the high kidney uptake and bladder
activity observed in the small animal PET studies suggests a renal route of excretion for these tracers. For
other organs including the heart, liver, and muscle the uptake was low to moderate for both enantiomers
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(
from 1 to 4.67 %ID/g during the study) with about twoꢀfold more uptake for (S)-[ F]8 than (R)-[ F]8 in
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most tissues. Bone activity was low and constant for (R)-[ F]8 (from 2.25 ± 0.73 %ID/g to 2.91 ± 0.86
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%
ID/g) while it was little higher and slowly increased over time for (S)-[ F]8 (from 2.69 ± 0.17 %ID/g to
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.33 ± 0.96 %ID/g). The increasing bone activity observed with (S)-[ F]8 may result from in vivo
defluorination but could potentially represent accumulation in the bone marrow. The tumor to bone ratio
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for (S)-[ F]8 at 60 min after injection was approximately 1.5 which is much higher than the ratio
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observed with the structurally related compound [ F]FMA, consistent with less in vivo defluorination
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due to the presence of the αꢀmethyl group in (S)-[ F]8.
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. Small animal PET/CT studies in mice with intracranial DBT tumors
The results from small animal PET studies in mice with intracranial DBT tumors are presented as
time activity curves (TACs) in Figure 6. The results showing average standardized uptake values (SUVs)
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obtained with (S)-[ F]8 and (R)-[ F]8 showed a peak tumor uptake of radioactivity within 7 min p.i. for
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both enantiomers. After the initial few min, which is dominated by flow, the tumor uptake of (S)-[ F]8
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was higher than (R)-[ F]8. Furthermore, the tumor uptake of (S)-[ F]8 was relatively constant during the
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study with only 10% washout whereas the tumor uptake with (R)-[ F]8 decreased by approximately 50%
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over the course of the study. At 45ꢀ60 min p.i., the average SUV was 0.83 ± 0.14 for Sꢀ[ F]8 compared to
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.49 ± 0.07 for (R)ꢀ[ F]8 (p < 0.05). In the normal brain, the average SUVs were similar for both
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enantiomers over time with slightly higher activity observed with (S)-[ F]8 compared to (R)-[ F]8; this
did not reach statistical significance. Overall, both enantiomers demonstrated good tumor imaging
properties, which was consistent with the biodistribution studies. Together, these data suggest that (S)-
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F]8 is the superior enantiomer for imaging due to higher tumor and brain uptakes at early and late time
points and better retention in the tumor over time.
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Figure 6: Time-activity curves of (S)-[ F]8 and (R)-[ F]8 uptake in intracranial DBT tumors and contralateral
normal brain. Mice were anesthetized with 1% isoflurane/oxygen for 0ꢀ60 min dynamic microPET/CT scans.
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Intravenous injection of 150ꢀ200 µCi (5.55ꢀ7.4 MBq) of (S)-[ F]8 was done on day 14 and (R)-[ F]8 on day 15
post unilateral tumor implantation. The same animals were used for imaging both tracers. The data are displayed as
average SUVs, and each time point represents the mean of 4 animals.
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In Figure 7, tumor to brain ratios of the two new enantiomers (S)-[ F]8 and (R)-[ F]8 measured
with small animal PET at 45ꢀ60 min after injection are compared with those obtained with the system L
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substrate (S)ꢀ[ F]FET and the system A substrate (R)-[ F]MeFAMP. The data show that (S)-[ F]8, and
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(
R)-[ F]8 had higher tumor to brain ratio compared to (S)-[ F]FET (p < 0.001 for both enantiomers) but
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lower than (R)ꢀ[ F]MeFAMP (p < 0.001 for both enantiomers). These findings suggest that (S)-[ F]8
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and (R)-[ F]8 could provide better tumor visualization and a larger dynamic range for monitoring
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response to therapy than (S)ꢀ[ F]FET. However, for (S)-[ F]8 and (R)-[ F]8 and even more for (R)ꢀ
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[
F]MeFAMP, the high tumor to brain ratios obtained are due primarily to low brain uptake which could
decrease visualization of nonꢀenhancing regions within gliomas. Conventional orthotopic models of high
grade gliomas have disrupted BBBs in tumor tissue which limits their predictive value for the
visualization of nonꢀenhancing tumor regions that are often encountered in human gliomas. Longer chain
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analogues of (S)-[ F]8 are expected to show enhanced system L transport and may prove superior to (S)-
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[
[
F]8 for imaging the nonꢀenhancing regions of brain tumors. Representative PET/CT images of (S)ꢀ
F]8 are shown in Figure 8. The amount of activity in the bone observed in the small animal PET
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studies was less than expected based on the biodistribution studies. The reason for this difference is not
clear but may be due to effects of anesthesia during the small animal PET studies.
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Figure 7: Tumor to brain ratios from small animal PET studies performed with (S)ꢀ[ F]8 and (R)ꢀ[ F]8 in the same
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animals (n= 4), and with (S)ꢀ[ F]FET and (R)ꢀ[ F]MeFAMP (n= 3) approximately 45ꢀ60 min after injection. SUVꢀ
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Tumor (S)ꢀ[ F]FAMPe = 0.83 ± 0.14, SUVꢀBrain (S)ꢀ[ F]FAMPe = 0.15 ± 0.03, SUVꢀTumor (R)ꢀ[ F]FAMPe = 0.49 ±
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.07, SUVꢀBrain (R)ꢀ[ F]FAMPe = 0.09 ± 0.02, SUVꢀTumor (S)ꢀ[ F]FET = 1.10 ± 0.09, SUVꢀBrain (S)ꢀ[ F]FET = 0.55 ±
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.05, SUVꢀTumor (R)ꢀ[ F]MeFAMP = 0.77 ± 0.23, SUVꢀBrain (R)ꢀ[ F]MeFAMP = 0.09 ± 0.02. Errors bars show SD. pꢀ
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Values represent comparisons of the tumor: brain ratios for both radiotracers, (S)ꢀ[ F]8 and (R)ꢀ[ F]8, with (S)ꢀ
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[
F]FET and with (R)ꢀ[ F]MeFAMP through 1ꢀway ANOVA with Tukey postꢀtests. * = p < 0.001.
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Figure 8: Representative (S)ꢀ[ F]8 small animal PET/CT (A) and PET (B) images of a mouse with an intracranial
DBT gliomas (red arrows) acquired 45ꢀ60 min after injection of tracer. Additional tumor tracking with less intense
tracer uptake is seen tracking along the skull. (C) Anterior and lateral maximum intensity projection (MIP) PET/CT
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images obtained 50-60 min after injection of (S)-[ F]8. The intracranial DBT tumor is designated by the red arrow
and is better seen on the lateral MIP due to the lack of obscuring salivary and nasopharyngeal activity.
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Conclusion
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Both enantiomers of a novel α,αꢀdisubstituted fluorinated amino acid, (S)- and (R)-[ F]8, were
prepared through efficient organic and radiosyntheses. Both tracers have promising imaging properties
18
with (S)-[ F]8 displaying superior properties compared to the (R)-enantiomer in the mouse DBT glioma
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model. In vitro cell uptake assays demonstrated that (S)- and (R)-[ F]8 enter DBT cells via multiple
transport systems including system L and other neutral amino acid transporters but not through system A
1
8
transport. Additionally, the uptake of (S)-[ F]8 was inhibited by Lꢀglutamine to a greater extent than the
(
R)-enantiomer. Biodistribution studies in a mouse subcutaneous DBT model of glioblastoma
1
8
demonstrated higher and more persistent tumor uptake with (S)-[ F]8 than with the (R)-enantiomer.
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Small animal PET imaging showed that (S)-[ F]8 had rapid and persistent intracranial tumor uptake with
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contralateral normal brain uptake greater than the system A tracer (R)-[ F]MeFAMP but less than the
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system L tracer (S)-[ F]FET. Overall (S)-[ F]8 is a promising new tracer for tumor imaging and may
serve as a lead compound for optimized system L and glutamine transport imaging agents.
Experimental Section
Materials and Instrumentation. All reagents and materials were purchased from commercially available
sources. Chemicals were purchased from Aldrich Chemicals Co. (Milwaukee, WI USA), Sigma Chemical
Co. (St Louis, Mo USA) and from Advanced Chemtech Co. (Louisville, KY USA), and solvents were
purchased from Aldrich Chemicals and Products (Pittsburg, PA USA). Chromatography was carried out
using silica gel 60 (0.040ꢀ0.063 mm) from EMD Millipore. Thinꢀlayer chromatography (TLC) analyses
were performed with 200µm UV254 silica gel backing on aluminum sheets (EMD Millipore USA). The
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TLC plates were revealed with ninhydrin and/or iodide stains. SepꢀPak C18 Plus Short Cartridge and
Oasis HLB Plus Light cartridge were purchased from Waters, Inc. (Milford, MA USA). Dionex OnGuard
II A cartridge was purchased from Fisher Scientific (Pittsburgh, PA). Melting points were measured with
an IA9100X1 Series digital melting point apparatus (ThermoFisher Scientific, USA) in capillary tubes
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and are uncorrected. H and C NMR spectra were recorded either on a 300 MHz or 400 MHz
NMR spectrometer (Varian/Agilent, Santa Clara, USA) maintained by the Washington University High
Resolution NMR Facility. Chemical shifts (δ values) are reported as parts per million (ppm) and coupling
values are reported in Hertz. Elemental analyses, performed by Atlantic Microlabs, Inc. (Norcross, GA
USA), were conducted on non-radioactive new intermediates synthesized during the multistep
synthesis and were within 0.4% of theoretical values with the exception of the non-radioactive
hydrochloric acid salt of the fluorinated amino acid (compound (R,S)-8). The purity of (R,S)-8 was
determined based on HPLC analysis and was higher than 95%. This non-radioactive compound was only
18
used as an HPLC standard for confirming the identity of the F-labeled target compounds but not for
18
biological studies. The purity of the final F-labeled amino acids used for biological studies was
measured using radiometric TLC and HPLC and met the requirements of 95% or greater purity. For the
compounds whose purity was determined by analytical HPLC, the Chirex 3126 Dꢀpenicillamine column
(
15 cm × 4.6 mm) was used with a solution of 85% 3 mM copper sulfate (CuSO ): 15% acetonitrile as
4
°
mobile phase, a flow rate of 1 mL/min, a detection at λ= 254 nm, a column temperature of 25 C. For
compound (R,S)ꢀ8, 10 µl of a 10.8 mM solution was injected. Mass spectra were run on a Bruker Maxis
QꢀToF mass spectrometer using highꢀresolution electron ionization and were carried out at the Chemistry
Department of Washington University in St Louis. Statistical analyses were performed using GraphPad
Prism software (GraphPad Software, La Jolla, CA) with pꢀvalues of 0.05 or less considered statistically
significant.
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Chemistry. Tert-butyl (S)-2-((diphenylmethylene)amino)propanoate (S)ꢀ1. Benzophenone imine (2.77
mL, 16.51 mmol) was added to a solution of Lꢀ(S)ꢀalanine tertꢀbutyl ester hydrochloride (3 g, 16.51
mmol) in 30 mL DCM. The mixture was stirred at rt for 2 h. A white precipitate was formed. The mixture
was diluted with water and two phases were separated. The aqueous phase was extracted with DCM (2 ×
30 mL). The combined organic layers were washed with brine (1 × 45 mL), dried over sodium sulfate
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(
Na SO ), filtered and finally concentrated to dryness under reduced pressure. (S)ꢀ1 was obtained as a
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colorless oil (94%) which was used without further purification.
1
H NMR (CDCl ): δ 1.40 (d, J= 6.7Hz, 3H), 1.45 (s, 9H), 4.03 (q, J= 6.7Hz, 1H), 7.18ꢀ7.20 (m, 2H),
3
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.30ꢀ7.48 (m, 6H), 7.63ꢀ7.65 (m, 2H). H NMR spectrum agrees with literature report.
13
C NMR (CDCl ): 19.3 (CH ), 28.1 (3CH ), 61.4 (CH), 80.8 (C), 127.8 (2CH), 128.1 (2CH), 128.6
3
3
3
(
2CH), 128.8 (2CH), 130.3 (2CH), 136.7 (C), 139.8 (C), 169.4 (C=N), 172.1 (C=O).
Tert-butyl (R,S)-2-((diphenylmethylene)amino)-2-methylpent-4-enoate (R,S)-2. Imine 1 (500 mg, 1.61
mmol) solubilized in 1 mL THF was added to a solution of LDA (1.77 mL of 2.0 M solution in THF,
heptane, ethylbenzene, 3.54 mmol) in 6 mL anhydrous THF at ꢀ78 °C under nitrogen. An orange enolate
solution resulted. The mixture was stirred at ꢀ78 °C for 1 h, and then the alkylating agent allyl iodide
(0.33 mL, 3.54 mmol) was added. The solution was stirred for overnight and allowed to return to rt. The
reaction was quenched with the addition of a saturated solution of ammonium chloride (NH Cl) (10 mL)
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and diluted with diethyl ether (Et O) (15 mL). Two phases were separated. The aqueous phase was
2
extracted with Et O (2 × 15 mL). The combined organic layers were washed with brine (1 × 20 mL),
2
dried over Na SO , filtered and concentrated to dryness under reduced pressure. The crude compound was
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purified on silica gel column chromatography eluted with 20% ethyl acetate in hexane to give (R,S)-2 as a
colorless oil (90%).
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H NMR (CDCl ): δ 1.24 (s, 3H), 1.30 (s, 9H), 2.59ꢀ2.69 (m, 2H), 5.10ꢀ5.14 (m, 2H), 5.96ꢀ6.07 (m, 1H),
3
7.20ꢀ7.37 (m, 8H), 7.54ꢀ7.56 (m, 2H).
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C NMR (CDCl ): 24.5 (CH ), 28.1 (3CH ), 48.1 (CH ), 66.7 (C), 80.9 (C), 117.9 (CH =), 128.0 (4CH),
3
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28.4 (CH), 128.7 (2CH), 128.9 (2CH), 129.9 (CH), 130.2 (CH), 138.6 (C), 141.8 (C), 167.1 (C=N),
73.6 (C=O).
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HRMS (ESI): m/z calculated for C H NO + H [M + H]+: 350.2115. Found: 350.2126.
23 27 2
Tert-butyl (R,S)-2-amino-2-methylpent-4-enoate (R,S)-3. Hydroxylamine hydrochloride (897 mg, 12.91
mmol) was added to a solution of 2 (1.29 g, 3.69 mmol) in 30 mL anhydrous methanol under nitrogen at
rt. After 1 hour, the reaction was complete. The mixture was then concentrated under reduced pressure.
Water (30mL) and DCM (60 mL) were added and the two phases were separated. The aqueous phase was
basified to pH=7.5 by using a saturated solution of potassium carbonate (K CO ), then extracted with
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DCM (2 × 40 mL). The combined organic layers were washed with brine (40 mL), dried over Na SO ,
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filtered, and concentrated to dryness under reduced pressure. The crude compound was purified on silica
gel column chromatography; it was eluted first with 20% ethyl acetate in hexane then with 10% methanol
in DCM to provide the free amine (R,S)-3 as a colorless oil (65%).
1
H NMR (CDCl ): δ 1.29 (s, 3H), 1.46 (s, 9H), 1.93 (bs, 2H), 2.21ꢀ2.26 (m, 1H), 2.46ꢀ2.52 (m, 1H), 5.11ꢀ
3
5.16 (m, 2H), 5.67ꢀ5.78 (m, 1H).
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C NMR (CDCl ): δ 26.5 (CH ), 28.1 (3CH ), 45.3 (CH ), 57.6 (C), 81.0 (C), 119.2 (CH =), 133.2
3
3
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(
CH=), 176.6 (C=O). NMR spectrums agree with literature report.
Tert-butyl (R,S)-2-((tert-butoxycarbonyl)amino)-2-methylpent-4-enoate (R,S)-4. Compound 3 (227 mg,
1.22 mmol) was solubilized in 10 mL THF, then diꢀtertꢀbutyl dicarbonate (532 mg, 2.44 mmol) was
added and the solution was stirred at rt overnight. Brine (20 mL) and DCM (40 mL) were added and the
two phases were separated. The aqueous phase was extracted with DCM (2 × 40 mL). The combined
organic layers were dried over Na SO , filtered, and concentrated to dryness under reduced pressure. The
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crude compound was purified on silica gel column chromatography; it was eluted first with 100% hexane
then with 10% ethyl acetate in hexane to provide the protected amine (R,S)-4 as a colorless oil (92%).
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H NMR (CDCl ): δ 1.42 (s, 9H), 1.45 (s, 9H), 1.50 (s, 3H), 2.48ꢀ2.53 (m, 1H), 2.77 (bs, 1H), 5.08ꢀ5.12
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(m, 2H), 5.28 (bs, 1H), 5.61ꢀ5.72 (m, 1H).
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C NMR (CDCl ): δ 23.4 (CH ), 28.0 (3CH ), 28.5 (3CH ), 41.3 (CH ), 59.3 (C), 81.8 (2C), 119.1
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(
CH =), 132.8 (CH=), 154.4 (C=O), 173.2 (C=O).
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HRMS (ESI): m/z calculated for C H NO + Na [M + Na]: 308.1832. Found: 308.1835.
15
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Elemental analysis calculated (%) for C H NO : C 63.13, H 9.54, N 4.91. Found: C 63.13, H 9.60, N
15 27
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4.99.
Tert-butyl (R,S)-2-((tert-butoxycarbonyl)amino)-5-hydroxy-2-methylpentanoate (R,S)-5. BoraneꢀTHF
solution (10.88 mL of 1.0 M solution, 10.88 mmol) was slowly added to a solution of 4 (840 mg, 2.94
mmol) in 60 mL anhydrous THF under nitrogen at 0 °C. The resulting solution was stirred for 2 hours at 0
°C. Then, still at 0 °C, a solution of 1 M sodium hydroxide (NaOH) (10.88 mL, 10.88 mmol) was added
slowly, followed by aqueous hydrogen peroxide 30% (4.60 mL, 40.26 mmol). The mixture was stirred
overnight and allowed to return to rt. Ethyl acetate (100 mL) and brine (100 mL) were added, and the two
layers were separated. The aqueous phase was extracted with ethyl acetate (2 × 150 mL). The combined
organic layers were dried over Na SO , filtered and concentrated to dryness under reduced pressure. The
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crude compound was purified on silica gel column chromatography eluted with 40% ethyl acetate in
hexane to give (R,S)-5 as a white solid (62%). mp: 94ꢀ95 °C
1
H NMR (CDCl ): δ 1.41 (s, 9H), 1.45 (s, 9H), 1.49 (s, 3H), 1.51ꢀ1.86 (m, 3H), 2.14 (bs, 1H), 3.61 (t, J=
3
6.3Hz, 2H), 5.43 (bs, 1H).
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C NMR (CDCl ): δ 23.7 (CH ), 27.5 (CH ), 28.0 (3CH ), 28.5 (3CH ), 32.9 (CH ), 59.6 (C), 62.7 (CH ),
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3
2
3
3
2
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81.9 (2C), 154.5 (C=O), 173.7 (C=O).
HRMS (ESI): m/z calculated for C H NO + Na [M + Na]: 326.1938. Found: 326.1941.
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Elemental analysis calculated (%) for C H NO : C 59.38, H 9.63, N 4.62. Found: C 59.39, H 9.72, N
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4.63.
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Tert-butyl (R,S)-2-((tert-butoxycarbonyl)amino)-2-methyl-5-(tosyloxy)pentanoate (R,S)-6. Sodium tertꢀ
butoxide (132mg, 1.37mmol) was added to a solution of the alcohol 5 (347 mg, 1.14 mmol) and pꢀ
toulenesulfonyl chloride (326 mg, 1.71 mmol) in 12 mL anhydrous DCM at 0 °C and under nitrogen.
After 5 min of stirring at 0 °C, the ice bath was removed, and the reaction was stirred overnight at rt. The
mixture was then concentrated under reduced pressure, and the crude product was purified on silica gel
column chromatography eluted with 10% ethyl acetate in hexane to provide (R,S)-6 as a white solid
(40%). mp: 59ꢀ61 °C
1
H NMR (CDCl ): δ 1.38 (s, 9H), 1.42 (s, 12H), 1.47ꢀ1.65 (m, 2H), 1.74ꢀ2.00 (m, 1H), 2.04ꢀ2.10 (m, 1H),
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2.43 (s, 3H), 3.90ꢀ4.02 (m, 2H), 5.34 (bs, 1H), 7.32 (d, J= 8.4Hz, 2H), 7.75 (d, J= 8.4Hz, 2H).
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C NMR (CDCl ): δ 21.7 (CH ), 24.2 (CH ), 27.8 (3CH ), 28.4 (3CH ), 29.8 (CH ), 32.2 (CH ), 59.3 (C),
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0.4 (CH ), 82.1 (2C), 127.9 (2CH), 129.9 (2CH), 132.9 (C), 144.9 (C), 154.1 (C=O), 173.2 (C=O).
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HRMS (ESI): m/z calculated for C H NO S + Na [M + Na]: 480.2026. Found: 480.2032.
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Elemental analysis calculated (%) for C H NO S: C 57.75, H 7.71, N 3.06. Found: C 57.89, H 7.69, N
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3.06.
Tert-butyl (R,S)-2-((tert-butoxycarbonyl)amino)-5-fluoro-2-methylpentanoate (R,S)-7. Cesium fluoride
198 mg, 1.70 mmol) was added to a solution of the tosylate 6 (155 mg, 0.34 mmol) in 1.5 mL anhydrous
(
2ꢀmethylbutanꢀ2ꢀol. The mixture was stirred at 100 °C for 1 hour. Then, the reaction mixture was
triturated with ethyl ether to remove most of the ionic salts. The filtrate was concentrated under reduced
pressure and the crude product was purified on silica gel column chromatography and eluted with 8%
ethylacetate in hexane to provide (R,S)-7 as a white solid (34%). mp: 69ꢀ71 °C
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H NMR (CDCl ): δ 1.41 (s, 9H), 1.44 (s, 9H), 1.50 (3H), 1.52ꢀ1.82 (m, 2H), 1.84ꢀ1.92 (m, 1H), 2.20 (bs,
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H), 4.29ꢀ4.40 (m, 1H), 4.41ꢀ4.51 (m, 1H), 5.40 (bs, 1H).
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C NMR (CDCl ): δ 23.6 (CH ), 25.6 (d, J (C,F)= 20.2Hz, CH ), 27.9 (3CH ), 28.5 (3CH ), 32.2 (CH ),
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9.5 (C), 82.0 (2C), 83.8 (d, J (C,F)= 165.6Hz, CH F), 154.2 (C=O), 173.5 (C=O).
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HRMS (ESI): m/z calculated for C H FNO + Na [M + Na]: 328.1895. Found: 328.1897.
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Elemental analysis calculated (%) for C H FNO : C 58.99, H 9.24, N 4.59. Found: C 58.88, H 9.24, N
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.53.
(
R,S)-2-amino-5-fluoro-2-methylpentanoic acid hydrochloride (R,S)-8. The protected amino acid 7 (21
mg, 0.069 mmol) was stirred in 0.3ꢀ0.4 mL of 4 M hydrochloric acid (HCl) at 60 °C for 90 min. The
mixture was then concentrated under reduced pressure. The residue obtained was triturated in Et O to
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precipitate the desired compound. After filtration, product 8 was obtained as a white solid (78%). mp:
1
72ꢀ173 °C
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H NMR (D O): δ 1.60 (s, 3H), 1.64ꢀ2.14 (m, 4H), 4.46 (dt, J (H,F)= 47.0Hz, J (H,H)= 5.7Hz, 2H).
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C NMR (D O): δ 21.6 (CH ), 24.1 (d, J (C,F)= 20.1Hz, CH ), 32.6 (CH ), 59.8 (C), 83.9 (d, J (C,F)=
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3
2
2
1
66.8Hz, CH F), 174.1 (C).
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HRMS (ESI): m/z calculated for C H FNO ꢀ Cl [M ꢀ Cl ]: 150.0925. Found: 150.0926.
6
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Di-tert-butyl (R,S)-2-methylpyrrolidine-1,2-dicarboxylate, (R, S)-9. This compound is a side product of
reaction providing (R, S)-7. It was obtained as a white solid (56%). NMR analysis showed two sets of
signals corresponding to the two conformational isomers.
1
H NMR (CDCl ): δ 1.42 (s, 3.3H), 1.43 (s, 5.7H), 1.43 (s, 3.3H), 1.44 (s, 5.7H), 1.47 (s, 1.9H), 1.49(s,
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.1H), 1.74ꢀ1.92 (m, 3H), 2.02ꢀ2.16 (m, 1H), 3.43 (t, 0.7H, J= 6.5Hz), 3.47ꢀ3.58 (m, 1.3H).
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C NMR (CDCl ): δ 22.3, 22.7, 23.3, 23.5, 28.0, 28.1, 28.5, 28.6, 39.3, 40.9, 48.2, 48.2, 65.2, 65.7, 79.2,
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9.9, 80.4, 80.7, 153.9, 173.8, 173.9. NMR spectrums agree with literature report.
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(
R)- or (S)-2-((tert-butoxycarbonyl)amino)-2-methylpent-4-enoic acid, (R)-10 or (S)-10. The respective
starting material (S)ꢀ(ꢀ)ꢀαꢀallylalanine or (R)ꢀ(+)ꢀαꢀallylalanine (500 mg, 3.87 mmol) was suspended in
11 mL methanolꢀtriethylamineꢀ1 N NaOH (9/1/1: v/v/v) and diꢀtertꢀbutyl dicarbonate (1.69 g, 7.74 mmol)
was added in one portion. The reaction was stirred at rt 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 3 N HCl. 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
sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The respective product (R)ꢀ
10 or (S)ꢀ10 was obtained as a colorless oil (88 and 89% respectively) which was used without further
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purification.
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H NMR (CDCl ): δ 1.44 (s, 9H), 1.54 (s, 3H), 1.44 (s, 9H), 1.54 (s, 3H), 2.61ꢀ2.75 (bm, 2H), 5.14ꢀ5.18
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(
m, 2H), 5.68ꢀ5.78 (m, 1H), NH and COOH were not observed.
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C NMR (CDCl ): δ 23.3 (CH ), 28.4 (3CH ), 41.4 (CH ), 59.1 (C), 77.4 (C), 120.1 (CH ), 132.07 (CH),
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55.4(C), 178.2 (C). NMR spectrums agree with literature report.
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Radiochemistry. [ F]ꢀfluoride was produced using an RDSꢀ11 cyclotron in the Washington University
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Cyclotron Facility. [ F]fluoride was produced from [ O]water and the O(p,n) F reaction. Typical
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radiosyntheses used approximately 50 mCi of [ F]fluoride. First, the [ F]fluoride was dried by
azeotropic distillation using acetonitrile (2 × 1 mL then 2 × 0.5 mL) in the presence of potassium
carbonate (0.5 mg in 25 µL water) and Kryptofix 2.2.2 (K2.2.2) (3 mg in 50 µL anhydrous acetonitrile)
between 96 and 105 °C under a flow of nitrogen. Then, a solution of the respective tosylate precursor (S)-
6
or (R)-6 (5 mg in 500 µL of anhydrous 2ꢀmethylbutanꢀ2ꢀol) was added, and the mixture was heated for
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0 min between 96 and 105 °C in a sealed vessel. Incorporation of [ F]fluoride was estimated using
radiometric thin layer chromatography (radioꢀTLC), the elution was done in 100% ethyl acetate. Then, ~
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