[18F](2S,4S)-4-(3-Fluoropropyl)glutamine as a tumor imaging agent

Article abstract of DOI:10.1021/mp500236y

Although the growth and proliferation of most tumors is fueled by glucose, some tumors are more likely to metabolize glutamine. In particular, tumor cells with the upregulated c-Myc gene are generally reprogrammed to utilize glutamine. We have developed new 3-fluoropropyl analogs of glutamine, namely [¹⁸F](2S,4R)- and [¹⁸F](2S,4S)-4-(3-fluoropropyl)glutamine, 3 and 4, to be used as probes for studying glutamine metabolism in these tumor cells. Optically pure isomers labeled with ¹⁸F and ¹⁹F (2S,4S) and (2S,4R)-4-(3-fluoropropyl)glutamine were synthesized via different routes and isolated in high radiochemical purity (>95%). Cell uptake studies of both isomers showed that they were taken up efficiently by 9L tumor cells with a steady increase over a time frame of 120 min. At 120 min, their uptake was approximately two times higher than that of L-[³H]glutamine ([³H]Gln). These in vitro cell uptake studies suggested that the new probes are potential tumor imaging agents. Yet, the lower chemical yield of the precursor for 3, as well as the low radiochemical yield for 3, limits the availability of [¹⁸F](2S,4R)-4-(3-fluoropropyl)glutamine, 3. We, therefore, focused on [¹⁸F](2S,4S)-4-(3-fluoropropyl)glutamine, 4. The in vitro cell uptake studies suggested that the new probe, [¹⁸F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, is most sensitive to the LAT transport system, followed by System N and ASC transporters. A dualisotope experiment using L-[³H]glutamine and the new probe showed that the uptake of [³H]Gln into 9L cells was highly associated with macromolecules (>90%), whereas the [¹⁸F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, was not (<10%). This suggests a different mechanism of retention. In vivo PET imaging studies demonstrated tumor-specific uptake in rats bearing 9L xenographs with an excellent tumor to muscle ratio (maximum of ～8 at 40 min). [¹⁸F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, may be useful for testing tumors that may metabolize glutamine related amino acids.

Full text of DOI:10.1021/mp500236y

Article
pubs.acs.org/molecularpharmaceutics
[18F](2S,4S)‑4-(3-Fluoropropyl)glutamine as a Tumor Imaging Agent
Zehui Wu,†,§ Zhihao Zha,†,§ Genxun Li,† Brian P. Lieberman,† Seok Rye Choi,† Karl Ploessl,†
and Hank F. Kung*,†,‡
‡
†Departments of Radiology and Pharmacology, University of Pennsylvania, 3700 Market Street, Philadelphia, Pennsylvania 19104,
United States
ABSTRACT: Although the growth and proliferation of most
tumors is fueled by glucose, some tumors are more likely to
metabolize glutamine. In particular, tumor cells with the
upregulated c-Myc gene are generally reprogrammed to utilize
glutamine. We have developed new 3-fluoropropyl analogs of
glutamine, namely [18F](2S,4R)- and [18F](2S,4S)-4-(3-
fluoropropyl)glutamine, 3 and 4, to be used as probes for
studying glutamine metabolism in these tumor cells. Optically
pure isomers labeled with 18F and 19F (2S,4S) and (2S,4R)-4-
(3-fluoropropyl)glutamine were synthesized via different
routes and isolated in high radiochemical purity (≥95%).
Cell uptake studies of both isomers showed that they were
taken up efficiently by 9L tumor cells with a steady increase over a time frame of 120 min. At 120 min, their uptake was
approximately two times higher than that of L-[3H]glutamine ([3H]Gln). These in vitro cell uptake studies suggested that the
new probes are potential tumor imaging agents. Yet, the lower chemical yield of the precursor for 3, as well as the low
radiochemical yield for 3, limits the availability of [18F](2S,4R)-4-(3-fluoropropyl)glutamine, 3. We, therefore, focused on
[18F](2S,4S)-4-(3-fluoropropyl)glutamine, 4. The in vitro cell uptake studies suggested that the new probe, [18F](2S,4S)-4-(3-
fluoropropyl)glutamine, 4, is most sensitive to the LAT transport system, followed by System N and ASC transporters. A dual-
isotope experiment using L-[3H]glutamine and the new probe showed that the uptake of [3H]Gln into 9L cells was highly
associated with macromolecules (>90%), whereas the [18F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, was not (<10%). This
suggests a different mechanism of retention. In vivo PET imaging studies demonstrated tumor-specific uptake in rats bearing 9L
xenographs with an excellent tumor to muscle ratio (maximum of ∼8 at 40 min). [18F](2S,4S)-4-(3-fluoropropyl)glutamine, 4,
may be useful for testing tumors that may metabolize glutamine related amino acids.
KEYWORDS: cancer, metabolism, PET imaging, glutamine and radiolabeling
by producing ammonium in the kidneys. Enhanced glutamine
utilization in cancers due to changes in the expression of
oncogenic signaling pathways can lead to glutaminolysis. In
these cases, blocking glutamine synthetic pathways may lead to
tumor cell death.10 Glutamine imaging agents may be useful for
testing the therapeutic efficacy of antitumor agents aimed at
reducing glutamine metabolism in tumors.
In order to study glutamine metabolizing tumors, we
previously prepared and tested L-5-[11C]glutamine.7 In tumor
cell uptake studies, the maximum uptake of L-5-[11C]glutamine
reached 17.9 and 22.5% uptake/100 μg protein at 60 min in 9L
and SF188 tumor cells, respectively. At 30 min after incubation,
>30% of the activity appeared to be incorporated into cellular
proteins. Dynamic small animal PET studies in rats bearing
xenografts of 9L tumor and in transgenic mice bearing
INTRODUCTION
■
In the past two decades, the use of 2-[18F]fluoro-2-deoxy-D-
glucose (FDG) and positron emission tomography (PET) has
achieved widespread acceptance as an effective tool for
detecting cancers with high rates of glycolysis. It is generally
accepted that a high rate of glucose metabolism (Warburg
effect) is associated with changes in tumor-driven alternative
gene expression.1,2 However, despite the tremendous promise
of FDG-PET for detecting and monitoring tumor metabolism,
a significant portion of malignant tumors are not FDG-positive
and can be missed in a FDG-PET scan. Accordingly, there is a
clear and urgent need to develop additional metabolic tracers,
particularly for cancers with low FDG-uptake.
Recent reports suggest that metabolic reprogramming may
cause some cancers to switch their energy source from glucose
to glutamine.3−6 These tumors could be imaged with18F-
labeled glutamine tracers.7−9 Glutamine, which is found
circulating in the blood and is also concentrated in the skeletal
muscles (0.5−1 mmol/L), has various critical functions: as a
substrate for DNA and protein synthesis, a primary source of
fuel for cells lining the inside of the small intestine and rapidly
dividing immune cells, and as a regulator of acid−base balance
Special Issue: Positron Emission Tomography: State of the Art
Received: March 29, 2014
Revised: August 5, 2014
Accepted: August 5, 2014
Published: August 5, 2014
© 2014 American Chemical Society
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Figure 1. Chemical structures of L-5-[11C]glutamine, 1, (11C]Gln), [18F](2S,4R)-4-fluoroglutamine, 2 ([18F](2S,4R)-4-FGln), [18F](2S,4R)-4-(3-
fluoropropyl)glutamines, 3, ([18F](2S,4R)-4-FPGln), and [18F](2S,4S)-4-(3-fluoropropyl)glutamines, 4, ([18F](2S,4S)-4-FPGln).
spontaneous mammary gland tumors showed prominent tumor
uptake and retention. The results suggested that L-5-[11C]-
glutamine would be useful for probing in vivo tumor
metabolism in glutaminolytic tumors.7
(Bruker DPX 200 spectrometer). Chemical shifts are reported
as δ values (parts per million) relative to remaining protons in
deuterated solvent. Coupling constants are reported in hertz.
The multiplicity is defined by s (singlet), d (doublet), t
(triplet), q (quartet), p (pentet), br (broad), or m (multiplet).
HPLC analyses were performed on an Aglient LC 1100 series.
High-resolution MS experiments were performed using an
Agilent Technologies LC/MSD TOF mass spectrometer.
Syntheses. Compounds 5−8 were synthesized according to
the procedures reported previously.9
Because 11C has a half-life of 20 min, it would not be practical
for most clinical settings. To make an imaging agent that would
be better for clinical use, we created an alternative metabolic
tracer labeled with 18F, which has a half-life of 110 min. In vitro
studies with [18F](2S,4R)-4-fluoroglutamine, 2 ([18F](2S,4R)-
4-FGln) showed that both 9L and SF188 tumor cells displayed
a high rate of glutamine uptake (maximum uptake ≈16% dose/
100 μg protein), and the radioactivity trapped inside the cell
was associated with the macromolecular fraction precipitated by
trichloroacetic acid (TCA). The cell uptake of [18F](2S,4R)-4-
FGln, 2, by SF188 cells is comparable to that of [3H]L-
glutamine but higher than that of FDG. Biodistribution and
PET imaging studies showed that [18F](2S,4R)-4-FGln, 2,
localized in tumors with a higher uptake than that of
surrounding muscle and liver tissues, suggesting that [18F]-
(2S,4R)-4-FGln, 2, is selectively taken up and trapped by the
tumor cells.8,9
(S)-tert-Butyl 2-(tert-butoxycarbonylamino)-4-(tosyloxy)
Butanoate (9). To a solution of compound 8 (554 mg, 2
mmol) in 10 mL of dry dichloromethane (DCM) was added
Et3N (1.4 mL, 10 mmol), 4-(dimethylamino)pyridine (DMAP,
24 mg, 0.2 mmol), and p-toluenesulfonyl chloride (TsCl, 764
mg, 4 mmol) at 0 °C. The mixture was stirred at room
temperature (rt) overnight. Ice-cold water (15 mL) was poured
into the reaction mixture and the mixture was extracted with
DCM (15 × 3 mL), the combined organic layer was dried with
magnesium sulfate (MgSO4) and was purified with flash
chromatography (FC, EtOAc/hexane = 2/8) to give 739.6 mg
One of the drawbacks of [18F](2S,4R)-4-FGln, 2, (and its
related optical isomers) is the radiolabeling reaction, which is
relatively difficult and prone to formation of stereoisomers due
a secondary fluorination reaction.9 To avoid this complication,
we have designed and tested [18F](2S,4R)-4-(3-fluoropropyl)-
glutamine, 3 ([18F](2S,4R)-4-FPGln), and [18F](2S,4S)-4-(3-
fluoropropyl)glutamine, 4 ([18F](2S,4S)-4-FPGln), as alter-
native probes for imaging glutamine metabolism (Figure 1).
The 4-(3-fluoropropyl)glutamines contain an extended propyl
group. This will make it easier for the SN2 fluorine substitution
with a good leaving group (OTs) used in the labeling
reaction. To preserve the amide functional group at the C5
position, we have synthesized two types of precursors suitable
for radiolabeling (fluoro for tosylate substitution reaction).
Reported herein is the preparation and in vitro and in vivo
studies of these glutamine analogs.
1
colorless oil 9 (yield: 86.1%). HNMR (200 MHz, CDCl3) δ:
1.39−1.54 (m, 18H), 2.08−2.21 (m, 2H), 2.47 (s, 3H), 4.06−
4.09 (m, 2H), 4.13−4.16 (m, 1H), 5.03−5.10 (m, 1H), 7.37 (d,
J = 7.8 Hz, 2H), 7.81 (d, J = 8.2 Hz, 2H). HRMS was calcd for
C20H32NO7S (M + H)+: 430.1899. Found: 430.1910.
(S)-tert-Butyl 2-(tert-butoxycarbonylamino)-4-cyanobuta-
noate (10). Sodium iodide (240 mg, 1.6 mmol) was added to a
solution of compound 9 (343 mg, 0.8 mmol) in 10 mL of
acetone (HPLC grade) at rt. The mixture was stirred at 60 °C
for 3 h. The solvent was then removed and the residue was
dissolved in 15 mL of DCM. The precipitated solid was filtered
out and filtrate was concentrated. N,N-Dimethylformamide
(DMF, 7 mL) and potassium cyanide (78 mg, 1.2 mmol) were
added to the residue. The mixture was stirred at rt overnight.
The reaction mixture was quenched with 25 mL of ethyl acetate
and was washed with H2O (10 × 3 mL). The organic layer was
dried over MgSO4 and filtered. The filtrate was concentrated,
and the residue was purified by FC (EtOAc/hexane = 2/8) to
EXPERIMENTAL SECTION
■
1
give 196.2 mg colorless oil 10 (yield: 86.3%). HNMR (200
General Information. All reagents used were commercial
products and were used without further purification unless
otherwise indicated. Boc-Glu(OBzl)−OH (Boc-L-glutamic acid
5-benzyl ester, 15) was purchased from Sigma-Aldrich. Flash
chromatography (FC) was performed using silica gel 60 (230−
MHz, CDCl3) δ: 1.39−1.52 (m, 18H), 1.90−2.04 (m, 1H),
2.20−2.30 (m, 1H), 2.40−2.46 (m, 2H), 4.20−4.30 (m, 1H),
5.12−5.22 (m, 1H). HRMS was calcd for C14H25N2O4 (M +
H)+: 302.2080. Found: 302.2078.
(2S,4S)-tert-Butyl 2-(tert-Butoxycarbonylamino)-4-cyano-
hept-6-enoate (11a) and (2S,4R)-tert-Butyl 2-(tert-Butoxy-
1
400 mesh, Sigma-Aldrich). H NMR spectra were obtained at
200 MHz and 13C NMR spectra were recorded at 50 MHz
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carbonylamino)-4-cyanohept-6-enoate (11b). LiHMDS
(lithium bis(trimethylsilyl)amide) (1.5 mL, 1 mol/L solution
in THF) was added to a three-necked 250 mL flask. The
mixture was cooled down to −78 °C with a dry ice−acetone
bath. Compound 10 (201 mg 0.67 mmol) in 3 mL of dry THF
solution was added dropwise over 30 min. After being stirred at
−78 °C for 2 h, allyl bromide (0.24 mL, 2.8 mmol) was added
dropwise over 15 min. The mixture was then stirred at −78 °C
for another 4 h. The reaction was quenched with 20 mL of
ethyl acetate and 15 mL of HCl (2 M) and extracted with ethyl
acetate (20 × 3 mL). The organic layer was dried over MgSO4
and filtered. The filtrate was concentrated, and the residue was
purified by FC (EtOAc/hexane = 2/8) to give 60.9 mg 11a
MHz, CDCl3) δ: 1.39−1.52 (m, 18H), 1.70−1.90 (m, 4H),
1.91−2.02 (m, 2H), 2.45 (s, 3H), 2.60−2.70 (m, 1H), 3.95−
4.15 (m, 2H), 4.16−4.25 (m, 1H), 5.10−5.19 (m, 1H), 7.37 (d,
J = 8.0 Hz, 2H), 7.80 (d, J = 8.2 Hz, 2H). HRMS was calcd for
C25H39N3O7S (M + NH4)+: 514.2587. Found: 514.2589.
Compound 13b was prepared from 12b (102 mg, 0.3 mmol),
Et3N (0.21 mL, 1.5 mmol), TsCl (115 mg, 0.6 mmol) and
DMAP (4 mg, 0.03 mmol), following the same procedure
described for compound 13a. Compound 13b: 100 mg (yield:
1
67.1%). HNMR (200 MHz, CDCl3) δ: 1.39−1.52 (m, 18H),
1.70−1.90 (m, 4H), 1.90−1.98 (m, 1H), 2.15−2.30 (m, 1H),
2.50 (s, 3H), 2.60−2.70 (m, 1H), 3.95−4.15 (m, 2H), 4.20−
4.35 (m, 1H); 5.10−5.19 (m, 1H), 7.37 (d, J = 8.2 Hz, 2H),
7.80 (d, J = 8.2 Hz, 2H). HRMS was calcd for C25H39N3O7S
(M + NH4)+: 514.2587. Found: 514.2589.
1
(yield: 28.0%) and 32.6 mg 11b (yield: 15.1%). 11a: HNMR
(200 MHz, CDCl3) δ: 1.39−1.52 (m, 18H), 1.90−2.24 (m,
2H), 2.44−2.62 (m, 2H), 2.71−2.85 (m, 1H), 4.35−4.45 (m,
1H), 5.22−5.27 (m, 1H), 5.30−5.36 (m, 2H), 5.70−5.91 (m,
1H). HRMS was calcd for C17H29N2O4 (M + H)+: 325.2127.
(2S,4S)-tert-Butyl 2-(tert-Butoxycarbonylamino)-4-cyano-
7-fluoroheptanoate (14a) and (2S,4S)-tert-Butyl 2-(tert-
Butoxycarbonylamino)-4-cyano-7-fluoroheptanoate (14b).
To a solution of tris(dimethylamino)sulfonium difluorotrime-
thylsilicate (TASF, 100 mg, 0.363 mmol) in 3 mL of DCM and
3 mL of DMF was added Et3N(HF)3 (0.021 mL) dropwise
followed by 13a (35 mg, 0.072 mmol) in 3 mL of DCM. The
mixture was then heated at 50 °C overnight. The reaction was
quenched by the addition of ice-cold water (5 mL) and diluted
with 50 mL of EtOAc, and then washed with H2O (15 mL × 2)
and brine (15 mL) and dried with MgSO4. The filtrate was
evaporated in vacuo and the residue was purified by FC
(EtOAc/hexanes = 2/8) to give 22 mg of colorless oil 14a
1
Found: 325.2125. 11b: HNMR (200 MHz, CDCl3) δ: 1.39−
1.52 (m, 18H), 1.70−1.90 (m, 1H), 2.05−2.25 (m, 1H), 2.3−
2.55 (m, 2H), 2.71−2.85 (m, 1H) 4.35−4.45 (m, 1H), 5.22−
5.36 (m, 3H), 5.70−5.91 (m, 1H). HRMS was calcd for
C17H29N2O4 (M + H)+: 325.2127. Found: 325.2125.
(2S,4S)-tert-Butyl 2-(tert-Butoxycarbonylamino)-4-cyano-
7-hydroxy-heptanoate (12a) and (2S,4R)-tert-Butyl 2-(tert-
Butoxycarbonylamino)-4-cyano-7-hydroxyheptanoate
(12b). To a solution of compound 11a (91 mg, 0.28 mmol) in
7 mL of THF was added 9-borabicyclo[3.3.1] nonane (9-BBN,
2.22 mL, 0.5 M solution in THF) dropwise at 0 °C. After being
stirred at 0 °C for 1 h, the reaction mixture was moved to rt and
stirred for another 48 h. The mixture was then cooled with an
ice-bath. H2O2 (0.31 mL of 30 wt % solution in H2O) and
NaOH (0.4 mL, 1 M) were added dropwise. The mixture was
stirred at rt for 30 min, diluted with 15 mL of H2O and
extracted with ethyl acetate. The organic layer was dried over
MgSO4 and filtered. The filtrate was concentrated, and the
residue was purified by FC (EtOAc/hexane = 1/1) to give 30.7
mg colorless oil 12a (yield: 32.1%). 1HNMR (200 MHz,
CDCl3) δ: 1.39−1.52 (m, 18H), 1.80−1.95 (m, 4H), 1.96−2.15
(m, 2H), 2.70−2.85 (m, 1H), 3.69−3.75(m, 2H), 4.20−4.35
(m, 1H), 5.20−5.35 (m, 1H). HRMS was calcd for
C17H30N2O5 (M + H)+: 343.2233. Found: 343.2258.
1
(yield: 88.8%). HNMR (200 MHz, CDCl3) δ: 1.39−1.52 (m,
18H), 1.70−1.90 (m, 4H), 1.91−2.10 (m, 2H), 2.12−2.26 (m,
1H), 2.66−2.80 (m, 1H), 4.20−4.32 (m, 1H), 4.51 (dt, J = 46.4
Hz, J = 6.4 Hz, 2H), 5.10−5.15 (m, 1H). HRMS was calcd for
C17H33FN3O4 (M + NH4)+: 362.2455. Found: 362.2485.
Compound 14b was prepared from 13b (80 mg, 0.16 mmol),
TASF (220 mg, 0.80 mmol), and Et3N(HF)3 (0.046 mL)
following the same procedure described for compound 14a.
1
Compound 14b: 39 mg (yield: 70.8%). HNMR (200 MHz,
CDCl3) δ: 1.39−1.52 (m, 18H), 1.70−1.92 (m, 4H), 1.91−2.30
(m, 2H), 2.70−2.85 (m, 1H), 4.20−4.35 (m, 1H), 4.51(dt, J =
48.2 Hz, J = 5.8 Hz, 2H), 5.10−5.22 (m, 1H). HRMS was calcd
for C17H33FN3O4 (M + NH4)+: 362.2455. Found: 362.2485.
(2S,4R)-2-Amino-4-carbamoyl-7-fluoroheptanoic Acid (3)
and (2S,4S)-2-Amino-4-carbamoyl-7-fluoroheptanoic Acid
(4). Compound 14a (37 mg, 0.11 mmol) in concentrated
HCl (1.2 mL) was stirred at rt for 6 h. The pH was then
adjusted to 7−8 with 5% ammonium solution. The neutralized
solution was submitted to a small column of Dowex 50WX8-
200 (H+ form, 10 g). The fraction containing product was
concentrated in vacuo and dried under high vacuum overnight
to afford the crude product as a white solid. It was further
purified by recrystallization from EtOH/H2O to provide 5.9 mg
Compound 12b was prepared from 11b (194 mg, 0.6 mmol),
9-BBN (4.8 mL, 0.5 M solution in THF), H2O2 (0.65 mL of 30
wt % solution in H2O), and NaOH (0.9 mL, 1 M), following
the same procedure described for compound 12a. Compound
1
12b: 91 mg (yield: 44.6%). HNMR (200 MHz, CDCl3) δ:
1.39−1.52 (m, 18H), 1.70−1.85 (m, 4H), 1.86−2.01 (m, 1H),
2.09−2.30 (m, 1H), 2.70−2.85 (m, 1H), 3.65−3.75(m, 2H),
4.25−4.40 (m, 1H), 5.10−5.25 (m, 1H). HRMS was calcd for
C17H30N2O5 (M + H)+: 343.2233. Found: 343.2258.
1
of white solid 4 (yield: 26.0%). HNMR (200 MHz, D2O) δ:
(2S,4S)-tert-Butyl 2-(tert-Butoxycarbonylamino)-4-cyano-
7-(tosyloxy)heptanoate (13a) and (2S,4R)-tert-Butyl 2-(tert-
Butoxycarbonylamino)-4-cyano-7-(tosyloxy)heptanoate
(13b). To a solution of compound 12a (137 mg, 0.4 mmol) in
7 mL of DCM was added Et3N (0.3 mL, 2.1 mmol) and DMAP
(5 mg, 0.04 mmol) at °C, followed by TsCl (153 mg, 0.8
mmol). The resulting mixture was stirred at 0 °C for 1 and 24 h
at rt. The reaction was quenched with 15 mL of water and
extracted with DCM (10 × 3 mL). The organic layer was dried
over MgSO4 and filtered. The filtrate was concentrated, and the
residue was purified by FC (EtOAc/hexane = 2/8) to give
1.49−1.56 (m, 3H), 1.57−1.80 (m, 1H), 1.80−1.95 (m, 2H),
2.51−2.59 (m, 1H), 3.45−3.52 (m, 1H), 4.51 (d.t, J = 46.4 Hz,
J = 6.4 Hz, 2H); 13CNMR (200 MHz, D2O) δ: 179.8, 174.0,
84.82 (d, J = 157.5 Hz), 53.0, 42.2, 33.4, 27.2 (d, J = 20 Hz),
28.1. HRMS was calcd for C8H16FN2O3 (M + NH4)+:
207.1145. Found: 207.1169.
Compound 3 was prepared from 14b (39 mg, 0.11 mmol)
and concentrated HCl (1.2 mL) following the same procedure
described for compound 4. Compound 3: 6.8 mg (yield: 30%).
1HNMR (200 MHz, D2O) δ: 1.49−1.70 (m, 4H), 1.70−1.90
(m, 1H), 2.01−2.15 (m, 1H), 2.40−2.50 (m, 1H), 3.42−3.56
1
180.2 mg of colorless oil 13a (yield: 90.7%). HNMR (200
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(m, 1H), 4.41 (dt, J = 47.4 Hz, J = 5.0 Hz, 2H). 13CNMR (50
MHz, D2O) δ: 179.9, 174.0, 84.82 (d, J = 157.5 Hz), 53.0, 42.1,
33.4, 27.4 (d, J = 30 Hz), 27.4. HRMS was calcd for
C8H16FN2O3 (M + NH4)+: 207.1145. Found: 207.1162.
(S)-5-Benzyl 1-tert-Butyl 2-(tert-Butoxycarbonylamino)-
pentanedioate (16). To a solution of Boc-Glu(OBzl)−OH,
15, (3.37 g, 10 mmol) in 20 mL of DCM was added tert-butyl
2,2,2-trichloroacetimidate (3.9 g, 18 mmol) in 20 mL of
cyclohexane dropwise. The mixture was stirred at rt for 5 min.
BF3·Et2O (0.12 mL, 1 mmol) was added dropwise. The
reaction mixture was stirred at rt for 4 h. The solid was filtered
off. The filtrate was evaporated in vacuo and the residue was
purified by FC (EtOAc/hexanes = 2/8) to give 3 g of white
2H), 2.53−2.60 (m, 1H), 3.30−3.53 (m, 2H), 3.64−3.87 (m,
2H), 4.11−4.21 (m, 1H), 4.53 (s, 1H), 4.89−4.93 (m, 1H),
5.15−5.31 (m, 2H), 7.36 (s, 5H). HRMS was calcd for
C29H46NO8 (M + H)+: 536.3223. Found: 536.3201.
(2S,4S)-5-tert-Butoxy-4-(tert-butoxycarbonylamino)-5-
oxo-2-(3-(tetrahydro-2H-pyran-2-yloxy)propyl)pentanoic
acid (20). A mixture of the ester 19 (750 mg, 1.4 mmol) and
10% Pd/C (90 mg) in absolute EtOH (15 mL) was stirred
under hydrogen overnight. This mixture was then filtered and
the filtrate was concentrated to give 620 mg of colorless oil 20
1
(yield: 99%). HNMR (200 MHz, CDCl3) δ: 1.45−1.47 (m,
18H), 1.50−1.70 (m, 6H), 1.75−1.80 (m, 2H), 1.82−2.00 (m,
4H), 2.51−2.58 (m, 1H), 3.41−3.51 (m, 2H), 3.80−3.95 (m,
2H), 4.22−4.29 (m, 1H), 4.59 (s, 1H), 4.05−5.01 (m, 1H).
HRMS was calcd for C22H40NO8 (M + H)+: 446.2754. Found:
446.2740.
1
solid 16 (yield: 76.3%). HNMR (200 MHz, CDCl3) δ: 1.44−
1.46 (m, 18H), 1.90−1.97 (m, 1H), 2.13−2.20 (m, 1H), 2.40−
2.50 (m, 2H), 4.20−4.23 (m, 1H), 5.06 (s, 1H), 5.13 (s, 2H),
7.36 (s, 5H). HRMS was calcd for C21H32NO6 (M + H)+:
394.2230. Found: 394.2241.
(2S,4S)-tert-Butyl 2-(tert-Butoxycarbonylamino)-7-hy-
droxy-4-(2,4,6-trimethoxybenzylcarbamoyl)heptanoate
(21). To a solution of compound 20 (534 mg, 1.2 mmol) in 2
mL of DCM and 2 mL of DMF was added Et3N (1.9 mmol,
0.26 mL), HOBt (1.44 mmol, 297 mg), 2,4,6-trimethoxybenzyl-
amine hydrochloride (370 mg, 1.58 mmol), and N,N-
dicyclohexylcarbodiimide (276 mg, 1.8 mmol) at 0 °C. The
mixture was stirred at rt for 24 h. A total of 30 mL of EtOAc
was added to the reaction mixture. The mixture was then
washed with citric acid (10% in H2O, 5 mL), H2O (5 mL × 2)
as well as brine (5 mL), dried over Na2SO4, and filtered. The
filtrate was concentrated to give 624 mg of oil. The residue was
dissolved in 4 mL of EtOH. Pyridinium p-toluenesulfonate (50
mg, 0.2 mmol) was then added. After heating at 50 °C for 4 h,
the solvent was evaporated in vacuo and the residue was
purified by FC (EtOAc/hexanes = 3/7) to give 203 mg of
colorless oil 21 (yield: 30.8%). 1HNMR (200 MHz, CDCl3) δ:
1.44−1.45 (m, 18H), 1.46−1.70 (m, 4H), 1.81−1.86 (m, 2H),
2.14−2.21 (m, 2H), 3.54−3.60 (m, 2H), 3.82 (s, 9H), 4.12−
4.18 (m, 1H), 4.28−4.37 (m, 1H), 4.53−4.62 (m, 1H), 5.04−
5.09 (m, 1H), 5.95 (s, 1H), 6.13 (s, 1H). HRMS was calcd for
C27H45N2O9 (M + H)+: 541.3125. Found: 541.3083.
(2S,4S)-1-Benzyl 5-tert-Butyl 2-Allyl-4-(tert-Butoxycarbo-
nylamino)-pentanedioate (17). LiHMDS solution (8.8 mL,
1 M in THF) was added to a three-necked 250 mL flask and
cooled down to −78 °C. Compound 16 (1.57 g, 4 mmol) was
dissolved in 6 mL of THF was then added dropwise over 30
min. The mixture was stirred at −78 °C for another 2 h. Allyl
bromide (2.4 g, 20 mmol, 1.72 mL) was added dropwise. The
mixture was then stirred at −78 °C for another 4 h. The
reaction was quenched with 20 mL of ethyl acetate and 15 mL
of HCl (2 M), the mixture was extracted with ethyl acetate (3 ×
25 mL). The organic layer was dried over MgSO4 and filtered.
The filtrate was concentrated, and the residue was purified by
FC (EtOAc/hexane = 2/8) to give 1 g of colorless oil 17 (yield:
1
58%). HNMR (200 MHz, CDCl3) δ: 1.45−1.47 (m, 18H),
1.94−2.01 (m, 2H), 2.37−2.44 (m, 2H), 2.59−2.69 (m, 1H),
4.18−4.30 (m, 1H), 5.02−5.04 (m, 1H), 5.07−5.22 (m, 4H),
5.61−5.82 (m, 1H), 7.38 (s, 5H). HRMS was calcd for
C24H36NO6 (M + H)+: 434.2543. Found: 434.2507.
(2S,4S)-1-Benzyl 5-tert-Butyl 4-(tert-Butoxycarbonylami-
no)-2-(3-hydroxypropyl)pentanedioate (18). To a solution
of compound 17 (860 mg, 2 mmol) in 7 mL of THF was added
9-BBN (8 mL, 0.5 M solution in THF) dropwise at 0 °C. After
stirring at 0 °C for 1 h, the reaction mixture was moved to rt
and stirred for another 48 h. The mixture was then cooled in an
ice-bath. H2O2 (2.2 mL of 30 wt % solution in H2O) and
NaOH (3 mL, 1 M) were added dropwise. The mixture was
stirred at rt for 30 min, diluted with of 15 mL H2O, and
extracted with ethyl acetate. The organic layer was dried over
MgSO4 and filtered. The filtrate was concentrated, and the
residue was purified by FC (EtOAc/hexane = 1/1) to give 700
(2S,4S)-tert-Butyl 2-(tert-Butoxycarbonylamino)-7-(tosy-
loxy)-4-(2,4,6-trimethoxybenzylcarbamoyl)heptanoate (22).
To a solution of compound 21 (200 mg, 0.37 mmol) in 10 mL
of DCM was added 4-(dimethylamino)pyridine (4.55 mg,
0.037 mmol), Et3N (147 mg, 1.11 mmol), and TsCl (105 mg,
0.55 mmol) at 0 °C. The mixture was stirred at rt overnight.
The reaction was then quenched with 15 mL of H2O and
extracted with ethyl acetate (15 mL × 3). The organic layer was
dried over MgSO4 and filtered. The filtrate was concentrated,
and the residue was purified by FC (EtOAc/hexane = 3/7) to
1
1
mg of colorless oil 18 (yield: 77.4%). HNMR (200 MHz,
give 209 mg of white solid 22 (yield: 81.7%). HNMR (200
CDCl3) δ: 1.43−1.45 (m, 18H), 1.75−1.86 (m, 4H), 1.89−1.93
(m, 2H), 2.49−2.63 (m, 1H), 3.59 (t, J = 6.4 Hz, 2H) 4.24−
4.27 (m, 1H), 5.04−5.1 (m, 3H), 7.36 (s, 5H). HRMS was
calcd for C24H38NO7 (M + H)+: 452.2648. Found: 452.2623.
(2S,4S)-1-Benzyl 5-tert-Butyl 4-(tert-Butoxycarbonylami-
no)-2-(3-(tetrahydro-2H-pyran-2-yloxy)propyl)-
pentanedioate (19). To a solution of compound 18 (530 mg,
1.2 mmol) in 10 mL of DCM was added 3,4-dihydro-2H-pyran
(225 μL, 2.4 mmol) and pyridinium p-toluenesulfonate (30 mg,
0.12 mmol). The mixture was stirred at rt for 3 h. The solvent
was evaporated in vacuo and the residue was purified by FC
(EtOAc/hexanes = 3/7) to give 578 mg of colorless oil 19
MHz, CDCl3) δ: 1.44−1.45 (m, 18H), 1.55−1.62 (m, 4H),
1.76−1.89 (m, 2H), 1.93−2.07 (m, 1H), 2.47 (s, 3H), 3.82−
3.84 (m, 9H), 3.94−4.04 (m, 2H), 4.04−4.10 (m, 1H), 4.28−
4.37 (m, 1H), 4.56−4.66 (m, 1H), 4.95−4.99 (m, 1H), 5.89−
5.94 (m, 1H), 6.14 (s, 1H) 7.35 (d, J = 8.0 Hz, 2H), 7.76 (d, J =
8.4 Hz, 2H). HRMS was calcd for C34H51N2O11S (M + H)+:
695.3214. Found: 695.3099.
(2S,4S)-1-Benzyl 5-tert-Butyl 4-(tert-Butoxycarbonylami-
no)-2-(3-(tosyloxy)propyl)pentanedioate (23). Compound
23 was prepared from 18 (0.370 g, 0.84 mmol), Et3N (424
mg, 4.20 mmol), TsCl (319 mg, 1.68 mmol), and DMAP (10.2
mg, 0.084 mmol) in 10 mL of DCM, with the same procedure
described for compound 22. Compound 23: 410 mg (yield:
1
(yield: 90%). HNMR (200 MHz, CDCl3) δ: 1.43−1.45 (m,
18H), 1.50−1.64 (m, 6H), 1.68−1.73 (m, 4H), 1.90−2.01 (m,
1
80.5%). HNMR (200 MHz, CDCl3) δ: 1.42(s, 9H), 1.45(s,
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9H), 1.48−1.60 (m, 4H), 1.77−1.95 (m, 2H), 2.45−2.51 (m,
4H), 3.97−3.99 (m, 2H), 4.08−4.18 (m, 1H), 4.90 (d, 1 H, J =
9.2 Hz), 5.10 (dd, 2H, J = 12.2 Hz, J = 22 Hz), 7.32−7.35 (m,
7H), 7.77 (d, 2H, J = 8.2 Hz). HRMS was calcd for
C31H44NO9S (M + H)+: 606.2737. Found: 606.2784.
The radiosynthesis was performed by a similar method as
described previously.9 Briefly, an activated SepPak Light QMA
Carb was loaded with [18F]fluoride (740 to 1480 MBq (20 to
40 mCi)) and eluted with 1 mL of 18-crown-6/KHCO3 (160
mg of 18-crown-6 in 18.6 mL of ACN/29 mg of KHCO3 in 3.4
mL of water). The solution was blown with argon until dry and
dried twice azeotropically with 1 mL of acetonitrile at 80 °C
under a flow of argon. The dried [18F]fluoride was cooled in an
ice bath and 5 mg of tosylate precursor (O-tosylate, 13a and
13b, respectively) was dissolved in 0.5 mL of DMSO and added
to the dried [18F]fluoride. The mixture was heated for 10 min at
110 °C in an oil bath. The mixture was then cooled in an ice
bath and added to 6 mL of water/1 mL of acetonitrile. The
mixture was loaded onto an activated Oasis HLB 3 cm3
cartridge, pushed through, and washed twice with 3 mL of
water. The desired radiolabeled compound was eluted with 1
mL of acetonitrile. The acetonitrile solution was blown until
dry. A mixture of 400 μL of TFA/100 of μL concentrated
H2SO4 was added and heated for 10 min at 120 °C in a capped
10 mL vial. TFA was removed under argon while still warm.
The reaction tube was then cooled in an ice bath. Water (1
mL) was slowly added and the mixture was neutralized by the
slow addition of a saturated Na2CO3 solution under heavy
shaking (∼1000 μL) (pH ∼ 8). The mixture was put through
an activated Oasis HLB 3 cm3, which was topped with ∼0.3 g of
Ag11-A8 resin. The radioactivity was eluted with phosphate
buffered saline (pH 7.0) in fractions of 0.5 mL volume to yield
the desired radioactive [18F](2S,4R)-4-FPGln, 3, and [18F]-
(2S,4S)-4-FPGln, 4, respectively.
(2S,4S)-1-Benzyl 5-tert-Butyl 4-(tert-Butoxycarbonylami-
no)-2-(3-fluoropropyl)pentanedioate (24). To a solution of
tris(dimethylamino)sulfonium difluorotrimethylsilicate (1.31 g,
4.75 mmol) in 5 mL of THF and 5 mL of DMF was added
Et3N(HF)3 (0.273 mL) dropwise followed by 23 (0.700 g, 1.16
mmol) in 5 mL of THF. The mixture was then heated at 45 °C
overnight. The reaction was quenched by the addition of ice-
cold water (5 mL) and diluted with 100 mL of EtOAc, then
washed with H2O (25 mL × 2) and brine (25 mL), and dried
with MgSO4. The filtrate was evaporated in vacuo and the
residue was purified by FC (EtOAc/hexanes = 2/8) to give
1
0.430 g of colorless oil 24 (yield: 82.0%). HNMR (200 MHz,
CDCl3) δ: 1.43 (s, 9H), 1.45 (s, 9H), 1.59−1.72 (m, 4H),
1.92−2.00 (m, 2H), 2.51−2.61 (m, 1H), 4.15−4.30 (m, 2H),
4.51−4.53 (m, 1H), 4.93 (d, 1H, J = 8.4 Hz), 5.13 (dd, 2H, J =
12.2 Hz, J = 23 Hz), 7.31−7.36 (m, 5H). HRMS was calcd for
C24H37FNO6 (M + H)+: 454.2605. Found: 454.2667.
(2S,4S)-5-tert-Butoxy-4-(tert-butoxycarbonylamino)-2-(3-
fluoropropyl)-5-oxopentanoic acid (25). A mixture of the
ester 24 (0.430 g, 0.95 mmol) and 10% Pd/C (0.100 g) in
absolute EtOH (10 mL) was stirred under hydrogen overnight.
This mixture was then filtered, and the filtrate was concentrated
under vacuum to give 0.345 g of colorless oil 25 (yield: 100%).
1HNMR (200 MHz, CDCl3) δ: 1.45(s, 9H), 1.47 (s, 9H),
1.61−1.83 (m, 4H), 1.91−2.03 (m, 2H), 2.43−2.62 (m, 1H),
4.17−4.39 (m, 2H), 4.51−4.67 (m, 1H), 5.06 (d, 1H, J = 9.2
Hz). HRMS was calcd for C17H31FNO6 (M + H)+: 364.2135.
Found: 364.2166.
The radiochemical and stereochemical purities were
determined by two different HPLC systems. System 1.
Column: Gemini 3u C18 150 × 4.6 mm, 3 μm. Mobile
phase (gradient) Solvent A: ACN. Solvent B: 0.1% FA.
Gradient, 1 mL/min: 0−3 min 95% B, 3−11 min 95%−5% B,
11−19 min 5%−95% B, 19−21 min 95% B. Retention times for
both 3 and 4 are ∼2.5 min. System 2. Column: Chirex 3126
(D)-penicillamine 250 × 4.6 mm, 4.6 μm. Mobile phase
(isocratic): 2 mM CuSO4 solution, 1 mL/min, column
temperature at 30 °C. The retention times of 3 and 4 are
14.6 and 20 min, respectively.
(2S,4S)-tert-Butyl 2-(tert-Butoxycarbonylamino)-7-fluoro-
4-(2,4,6-trimethoxybenzylcarbamoyl)heptanoate (26). To a
solution of 25 (0.345 g, 0.95 mmol), N-(3-(dimethylamino)-
propyl)-N-ethylcarbodiimide hydrochloride (0.258 g, 1.34
mmol) and 1-hydroxybenzotriazole hydrate (0.227 g, 1.34
mmol) was added triethylamine (0.485 g, 4.80 mmol) and
2,4,6-trimethoxybenzylamine hydrochloride (0.339 g, 1.45
mmol) at 0 °C. The solution was allowed to warm to rt.
After stirred overnight, the mixture was diluted with 150 mL to
EtOAc and washed with H2O (30 mL × 2) and brine (30 mL).
The organic layer was dried by MgSO4 and concentrated to
give an oil that was purified by FC (EtOAc/hexane = 1/1) to
Alternatively, TmobNH precursor, 22, was used under a
similar labeling condition as described above (18-crown-6/
KHCO3/ACN/80 °C/20 min). It was found that the
TmobNH intermediate, 26, was formed with a lower yield of
6.6
1.6%, radiochemical purity 98% (n = 3). The 18F
intermediate, [18F]26, displayed the same profile on the HPLC
as that of the “cold” compound. Deprotection was performed
with 500 μL of TFA at 40 °C for 8 min. Volatiles were removed
under argon while still warm. The residue was treated with 1
mL of phosphate buffered saline (PBS) and filtered through a
0.45 μ filter and washed with 0.1 mL of PBS (pH 7.0) to give a
crude dose. The solution was passed through an activated
cartridge (Oasis HLB 3 cm3). The solid-phase extraction was
further rinsed with 0.3 mL of PBS (pH 7.0) to yield
(2S,4S)[18F]4-FPGln, 4.
1
give 0.350 g of colorless oil 26 (yield: 64.5%). HNMR (200
MHz, CDCl3) δ: 1.42 (s, 9H), 1.45 (s, 9H), 1.60−1.74 (m,
4H), 1.88−1.98 (m, 2H), 2.29−2.48 (m, 1H), 3.82 (s, 6H),
4.02−4.14 (m, 1H), 4.25−4.40 (m, 2H), 4.49−4.68 (m, 2H),
5.02 (br s, 1H), 6.20 (s, 2H), 6.34 (br s, 1H). HRMS was calcd
for C27H44FN2O8 (M + H)+: 543.3082. Found: 543.3048.
Radiolabeling. [18F]Fluoride was purchased from IBA
Molecular (Somerset, NJ) as an [18O]enriched aqueous
solution of [18F]fluoride. Solid-phase extraction (SPE)
cartridges such as Sep-Pak QMA Light and Oasis HLB
cartridges were purchased from Waters (Milford, MA). High-
performance liquid chromatography (HPLC) was performed
on Agilent 1100 or 1200 series system with different HPLC
columns.
Cell Uptake Studies. 9L cells (ATCC, Manassas, VA) were
cultured in Dulbecco’s Modified Eagle’s Medium (DMEM,
GIBCO BRL, Grand Island, NY) supplemented with 10% fetal
bovine serum (Hyclone, Logan, UT) and 1% 100 units/mL
penicillin, 100 μg/mL streptomycin. The cells were maintained
in T-75 culture flasks under humidified incubator conditions
(37 °C, 5% CO2) and were routinely passaged at confluence.
[3H]Gln was purchased from PerkinElmer (Waltham, MA)
with >97% radiochemical purity and 1.11−2.22 TBq/mmol
specific activity.
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a
Scheme 1. Production of [18F](2S,4R)-4-(3-fluoropropyl)glutamine
a
Reagents and reaction conditions: (a) tert-butyl 2,2,2-trichloroacetimidate (TBTA), BF3·Et2O, DCM, cyclohexane, rt, overnight; (b) Pd/C, H2,
MeOH, rt, overnight; (c) ECF, NaBH4, THF, H2O, −15−0 °C, 4 h; (d) TsCl, Et3N, DMAP, DCM, rt, overnight; (e) NaI, KCN, DMF, rt,
overnight;( f) LiHMDS, Allyl bromide, THF, −78 °C, 4 h;( g) 9-BBN, H2O2, NaOH, 0 °C - rt, 48 h; (h) TASF, Et3N(HF)3, DCM, DMF, 50 °C,
overnight; (i) HCl, rt, 6 h.
Tumor cells were plated in 12 well plates 24 h prior to
studies. On the day of the experiment, the culture media was
aspirated and the cells were washed three times with warm PBS
(containing 0.90 mM of Ca2+ and 1.05 mM of Mg2+).
[18F](2S,4R)-4-FPGln, 3 (370 kBq) and L-[3,4-3H(N)]-
glutamine ([3H]Gln) (37 kBq) were mixed in PBS (with
Ca2+ and Mg2+) solution and then added to each well. The
same procedure was performed with [18F](2S,4S)-4-FPGln, 4
and [3H]Gln. The cells were incubated at 37 °C for 5, 30, 60,
and 120 min. At the end of the incubation period, the PBS
solution containing the ligands was aspirated and the cells were
washed three times with 1 mL of ice cold PBS (without Ca2+
and Mg2+). After washing with ice-cold PBS, 350 μL of 0.1 N
NaOH was used to lyse the cells. The lysed cells were collected
onto filter paper and counted together with samples of the
incubation dose using a gamma counter (Packard Cobra). After
(Beckman LS 6500). A total of 100 μL of the cell lysate was
used to determine the protein concentration (Modified Lowry
Protein Assay). The data was normalized as percentage uptake
of initial dose (ID) relative to 100 μg of protein content (%
ID/100 μg of protein).
Protein Incorporation of [18F](2S,4S)-4-FPGln, 4, into
9L Tumor Cells. To test the in vivo cell incorporation, we
used 9L cells. Cells were plated (5 × 105 cells/well) on six-well
plates in culture media 24 h prior to the experiment. On the
day of experiment, the media was aspirated and the cells were
washed three times with 4 mL of warm PBS (containing 0.90
mM of Ca2+and 1.05 mM of Mg2+). To measure the extent of
protein incorporation of [18F](2S,4S)-4-FPGln, 4, protein
bound activity in 9L cells was determined at 30 and 120 min
after incubation. [18F](2S,4S)-4-FPGln, 4 (370 kBq) and L-
[3,4-3H(N)]-glutamine ([3H]Gln, 37 kBq) in 2 mL of PBS
were mixed in the incubation media.
3
24 h, H activity was counted using a scintillation counter
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a
Scheme 2. Production of [18F](2S,4S)-4-(3-fluoropropyl)glutamine
a
Reagents and conditions: (a) tert-butyl 2,2,2-trichloroacetimidate (TBTA), BF3·Et2O, DCM, cyclohexane, rt, overnight; (b) LiHMDS, Allyl
bromide, THF, −78 °C, 4 h;( c) 9-BBN, H2O2, NaOH, 0 °C−rt, 48 h; (d) DHP, PPTS, DCM, rt, 3 h; (e) Pd/C, H2, EtOH, rt, 6 h; (f) TmobNH2·
HCl, EDCI·HCl, HOBt, Et3N, DCM, DMF, rt, 24 h; (g) PPTS, EtOH, 50 °C, 4 h; (h) TsCl, Et3N, DMAP, DCM, rt, overnight; (i) TASF,
Et3N(HF)3, DCM, DMF, 50 °C, overnight.
To identify the ligand’s stability in supernatant after
precipitation with trichloroacetic acid (TCA), cells were
grown in 10 cm dishes and incubated with 1.8 MBq
[18F](2S,4S)-4-FPGln, 4, only. At the end of incubation, the
radioactive medium was removed, the cells were washed three
times with ice cold PBS without Ca2+ and Mg2+, treated with
0.25% trypsin, and resuspended in PBS. The samples were
centrifuged (18 000g, 3 min), the supernatant removed, and the
cells were suspended in 200 μL and 1% Triton-X 100 (Sigma,
St. Louis, MO). After vortexing, 800 μL of ice cold 15% TCA
was added to the solution. After precipitating for 10 min, the
cells were centrifuged again (18 000g, 3 min) and washed twice
with 15% ice cold TCA. The radioactivity of both gamma- and
beta-emitting isotopes was determined separately for the
supernatant and pellet. Protein incorporation was calculated
as a percentage of acid precipitable activity.
In Vitro Transport Characterization Studies (Inhib-
ition Studies). To characterize the transport of [18F](2S,4S)-
4-FPGln, 4, competitive inhibition studies were conducted
using the 9L cell line. The tracer was incubated at 37 °C for 30
min. The cells were processed as described above. Various
inhibitors were then added to the cells in concentrations
ranging from 0.1 to 5 mM in PBS solution. Selected inhibitors
included synthetic amino acid transport inhibitors such as N-
methyl-α-aminoisobutyric acid (MeAIB) for system A, and 2-
amino-bicylo[2.2.1] heptane-2-carboxylic acid (BCH) for
system L.11−13 Natural amino acids, such as L-serine and L-
glutamine, were also used as inhibitors, although they are not
specific for a particular amino acid transport system. The data
was compared in reference to uptake of [18F](2S,4S)-4-FPGln,
4, without any inhibitor in PBS solution at pH 7.4.
Biodistribution Studies in Rats Bearing 9L Tumors.
Studies of the in vivo distribution of [18F](2S,4S)-4-FPGln, 4,
were performed in Fischer (F344) rats bearing 9L tumors as
reported previously.8 F344 rats were purchased from Charles
River Laboratories (Malvern, PA). 9L tumor cells (∼106) in
PBS (0.2 mL) were injected subcutaneously into the lower right
flank of the rat. The tumors took 12−15 days to reach
appropriate size (1 cm diameter). All animals were fasted for
12−18 h prior to the study. Six rats per group were used for the
biodistribution study. The rats were anesthetized with
isoflurane (2−3%) and 0.2 mL of saline solution containing
25 μCi of the ligand was injected intravenously. The rats were
sacrificed at 30 and 60 min postinjection by cardiac excision
while under isoflurane anesthesia. The organs of interest were
removed, weighed, and the radioactivity was counted with a
gamma counter (Packard Cobra). The percent dose per gram
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Figure 2. Comparison of X-ray crystallography structures for (2S,4R)-4-fluoro-glutamine, (2S,4R)-4-FGln, 2,9 and (2S,4S)-4-(3-fluoropropyl)-
glutamine, (2S,4S)-4-FPGln, 4. ORTEP drawing of the title compounds were shown with 30% probability thermal ellipsoids. (Crystal structure,
(2S,4S)-4-FPGln, 4, was submitted to the Cambridge Crystallographic Data Centre (CCDC 991692).
was calculated by a comparison of the tissue activity counts to
counts of 1% of the initial dose.
were separated and purified by flash chromatography. They
were converted to the corresponding alcohols, 12a and 12b,
and following the treatment with tosyl chloride to the O-
tosylated 13a and 13b in good yields. The O-tosylated 13a and
13b were treated with TASF, Et3N(HF)3, DCM, DMF, 50 °C,
overnight to give the desired 14a and 14b, in good yields.
Optimization of the fluorination reaction condition using TASF
and Et3N(HF)3, as the reagents was reported previously for the
preparation of 4-fluoroglutamine.9 Deprotection using hydro-
chloric acid at room temperature produced the final end
products, [18F](2S,4R)-4-(3-fluoropropyl)glutamine, 3, and
[18F](2S,4S)-4-(3-fluoropropyl)glutamine, 4. The O-tosylated
13a and 13b were also successfully used for the radiolabeling
reaction (see Discussion below).
Small Animal Imaging Studies. Dynamic small animal
PET (APET) imaging studies were conducted with [18F]-
(2S,4R)-4-FGln, 2, and [18F](2S,4S)-4-FPGln, 4 similar to that
reported previously.8 All scans were performed on a dedicated
animal PET scanner (Mosaic by Phillips) that has a field of
view of 11.5 cm. F344 rats with 9L tumors were used for the
imaging studies. A total of 22−37 MBq of activity was injected
intravenously via the lateral tail vein. All animals were sedated
with isoflurane anesthesia (2−3%, 1 L/min oxygen) and were
then placed on a heating pad in order to maintain body
temperature throughout the procedure. The animals were
visually monitored for breathing and any other signs of distress
throughout the entire imaging period. The data acquisition
began after an intravenous injection of the tracer. All scans were
conducted over a period of 120 min (dynamic, 5 min/frame).
The frames were reconstructed and then analyzed with AMIDE
imaging analysis software.
The second method introduced N-Tmob protected
precursors (as a protecting group to preserve the amide) for
radiolabeling and deprotection. Previously, we have tested for
the preparation of N-Tmob protected precursors for making
isomers of 4-fluoroglutamine (4-FGln).9 We successfully
developed 18F labeling using this precursor under different
labeling conditions. We wanted to extend the same method to
the synthesis and labeling of [18F](2S,4S)-4-(3-fluoropropyl)-
glutamine, 4. To achieve this, we started with commercially
available, Boc-Glu(OBzl)−OH, 15. After treating with tert-
butyl 2,2,2-trichloroacetimidate/BF3·Et2O at room temper-
ature, it gave the t-Bu ester, 16. Using the same dianionic
allylation reactions of amino acid derivatives,14 the reaction
preferentially produced the protected (2S,4S)-4-allyl-glutamate
(in 58% yield). It is interesting to note that the reaction led to
the (2S,4S) isomer only. The allyl group was converted to
alcohol, 18, by 9-BBN/H2O2/NaOH in 0 °C. The alcohol was
protected by THP, and the O-benzyl ester was hydrolyzed and
the acid, 20, was transformed to Tmob-protected amide, 21.
The alcohol, 21, was treated with tosyl chloride to the O-
RESULTS
■
Synthesis. In order to produce [18F](2S,4R)-4-(3-
fluoropropyl)glutamine, 3, ([18F](2S,4R)-4-FPGln) and [18F]-
(2S,4S)-4-(3-fluoropropyl)glutamine, 4 ([18F](2S,4S)-4-
FPGln), we employed two different schemes (Schemes 1 and
2) for preparation of nonradioactive “cold” compounds (3 and
4) and the cyanide and -OTs precursors for radiolabeling. One
approach was to prepare the corresponding protected 4-cyanide
derivatives, which led to the formation of the desired final
products. Commercially purchased Boc-Asp(OBzl)−OH, 5,
was treated with tert-butyl 2,2,2-trichloroacetimidate/BF3·Et2O
at room temperature to give the t-BuO- ester, 6; the BnO- ester
group was converted to the acid, 7, by Pd/C catalyzed
hydrogenation. The aspartic acid, 7, was carefully reduced with
NaBH4 in THF/water at −15 to 0 °C to the corresponding
alcohol, 8. The alcohol group was successfully converted to the
cyanide, 10, through the -OTs intermediate, 9. The cyanide
derivative, 10, was treated with LiHMDS and allyl bromide at
−78 °C to give (2S,4S)-tert-butyl 2-(tert-butoxycarbonylami-
no)-4-cyanohept-6-enoate (11a) and (2S,4R)-tert-butyl 2-(tert-
butoxycarbonylamino)-4-cyanohept-6-enoate (11b) (11a to
11b ratio of 2:1 in 28% and 15% isolated yields). A similar
reaction was reported previously for preparation of allyl
derivatives of aspartate using the dianionic allylation reactions
of amino acid derivatives.14 The allyl derivatives, 11a and 11b,
tosylated, 22, which is a suitable precursor for a radioactive 18
F
labeling reaction. In order to provide an authentic sample, a
cold standard, for the first step of the radioactive 18F labeling
reaction, we also prepared compound, 26.
To further confirm the chemical structure, a slow evaporating
recrystallization method provided excellent crystals of “cold”
(2S,4S)-4-FPGln, 4 and the X-ray crystallographic analysis data
added support to the structure assignment (Figure 2). The
optically pure (2S,4S)-4-FPGln, 4, has never been prepared and
presented before. In the X-ray crystallographic structures of
(2S,4R)4F-Gln, 2, and (2S,4S)-4-FPGln, 4, the amino acid
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a
Scheme 3. Procedures for radiolabeling of [18F](2S,4R)-4-FPGln, 3, and [18F](2S,4S)-4-FPGln, 4
a
Reagent and conditions: (a) 18-crown-6/KHCO3/18F−/DMSO, 110 °C, 10 min; (b) 400 μL of TFA/100 μL of H2SO4 concentrated/120 °C, 10
min; (c) 18-crown-6/KHCO3/18F−/ACN, 80 °C, 20 min; (d) 500 μL of TFA/40 °C, 8 min.
Figure 3. HPLC profiles of [18F](2S,4S)-4-FPGln, 4. Profiles A and B were obtained using a gradient system: Gemini 3u C18 150 × 4.6 mm;
Gradient, 1 mL/min; Solvent A: ACN, solvent B: 0.1% aqueous formic acid: 0−3 min 95% B, 3−11 min 95%−5% B, 11−19 min 5%−95% B, 19−21
min 95% B. Profiles C and D were obtained using a chiral column and isocratic system: Chirex 3126 (D)-penicillamine 250 × 4.6 mm, 2 mM CuSO4
solution, 1 mL/min, column temperature at 30 °C. The radioactive peak displayed the same retention time as that of the “cold” standard under the
same HPLC conditions.
groups on the right side of the molecules were comparable,
whereas the amide group on the left appeared to be varied and
different from each other. The results reported in Figure 2
firmly establish the configuration, which may facilitate future
use of other 4-fluoroglutamine isomers for biological and
medical applications.
3 cm3 cartridge). The intermediate was eluted from this
cartridge and treated with H2SO4/TFA at 120 °C for 10 min.
The crude labeled products were cooled to room temperature
and neutralized with a saturated Na2CO3 solution. The mixture
was passed through an Oasis HLB 3 cm3 cartridge topped with
Ag11-A8 resin. The cartridge was eluted with phosphate
buffered saline (pH 7.0) to give the desired radioactive
[18F](2S,4R)-4-FPGln, 3, and [18F](2S,4S)-4-FPGln, 4, re-
spectively (Scheme 3). The purity was measured with reversed-
phase HPLC (radiochemical purity) and chiral HPLC (optical
purity). (See Figure 3.) The decay-corrected radiochemical
yield was 6.2 3.9%, radiochemical purity 91.5 1.5%, optical
purity >99%, n = 2 (for 3) and 25.2 2.3%, RCP 92.8 2.6%,
optical purity >99%, n = 5 (for 4). It is important to note that
radiolabeling of these two seemingly close analogs showed very
different yields. We noted the disparity in radiolabeling yields,
but we do not have a simple explanation for this phenomenon.
Radiolabeling for [18F](2S,4R)-4-(3-Fluoropropyl)-
glutamine, 3 ([18F](2S,4R)-4-FPGln, 3), and [18F](2S,4S)-
4-(3-Fluoropropyl)glutamine, 4 ([18F](2S,4S)-4-FPGln, 4).
Radiolabeling of the desired [18F](2S,4R)-4-FPGln, 3, and
[18F](2S,4S)-4-FPGln, 4, was achieved by methods in Scheme
3. The preparation can be accomplished by using the O-
tosylated cyanide derivatives, 13a or 13b, or the O-tosylated
TmobNH- protected precursor, 22. The substitution of O-Ts
with [18F]fluoride using O-tosylated cyanide derivatives, 13a or
13b, was performed with 18-crown-6/KHCO3 in DMSO at 110
°C for 10 min, followed by a solid-phase extraction (Oasis HLB
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Additional studies may be needed to investigate the optical
preferences in the substitution of O-Ts with [18F]fluoride. To
improve the radiolabeling reaction for the more promising
[18F](2S,4S)-4-FPGln, 4, we made the effort to use a different
O-tosylated TmobNH- protected precursor, 22. A similar 4-O-
tosylated TmobNH-protected precursor was successfully
employed for substitution of O-Ts with [18F]fluoride using
similar reaction conditions to give the desired [18F](2S,4R)-4-
FGln, 2, in good radiochemical yields (30−40%).9 However,
much to our surprise, the radiochemical yield for precursor, 22,
gave a lower labeling yield. The decay corrected radiochemical
yield was 2.7 0.9%, n = 3.
In Vitro Cell Uptake and Inhibition Study in 9L Tumor
Cells. In order to test the specificity of this radiotracer, in vitro
cell uptake and inhibition studies were performed in 9L cells.
Both [18F](2S,4R)-4-FPGln, 3, and [18F](2S,4S)-4-FPGln, 4,
displayed excellent uptake in the 9L tumor cells in vitro. At all
time points studied (5 to 120 min), both tracers displayed very
similar values (Figure 4). It appeared that the stereoisomers 4S
the [3H]Gln (>90%) was incorporated into macromolecules,
whereas the glutamine analog, [18F](2S,4S)-4-FPGln, 4,
remained predominantly in the supernatant (no incorporation).
On the basis of the protein incorporation data above (Figure
6), [18F](2S,4S)-4-FPGln, 4, behaved very differently from that
of [3H]Gln. It is reasonable to conclude that the new probe,
[18F](2S,4S)-4-FPGln, 4, is not associated with intracellular
macromolecules, and thus, it is less likely to measure the
intracellular metabolism associated with glutamine metabolism.
Biodistribution in F344 Rats Bearing 9L Tumor.
Biodistribution studies of [18F](2S,4S)-4-FPGln, 4, were
conducted in F344 rats (125−149 g, n = 4) bearing 9L tumors
on their thigh. This is a well-established animal model that
resembles typical human glioblastomas in clinical settings.15
Rats were sacrificed at 30 and 60 min postinjection by cardiac
excision while under isoflurane anesthesia. [18F](2S,4S)-4-
FPGln, 4, showed respectable uptake within the 9L tumors,
displaying 0.83% dose/g uptake at 30 min post injection.
Tumor uptake and retention slowly washed out of the 9L
tumor to 0.60% dose/g. At 30 min, tumor-to-background
(tumor-to-muscle, tumor-to-blood, and tumor-to-brain) ratios
of [18F](2S,4S)-4-FPGln, 4, were 6.91, 1.45, and 5.53,
respectively. The highest uptake of [18F](2S,4S)-4-FPGln, 4,
was found in the pancreas. High pancreatic uptake is consistent
with the fact that amino acids are precursors for digestive
enzymes actively produced in the pancreas. Low bone (femur)
uptake was observed at 30 min (0.53% dose/g) and it stayed at
that value at 60 min post injection.
Preliminary PET imaging studies of [18F](2S,4S)-4-FPGln, 4,
in rats with 9L tumors showed that the probe was clearly taken
up by the tumors (n = 3) (Figure 7). To further investigate the
tumor uptake, dynamic small animal PET studies using one rat
bearing two 9L tumors were carried out on two different days
with either [18F](2S,4S)-4-FPGln, 4, or [18F](2S,4R)-4-FGln, 2.
The direct comparison study using the same animal can avoid
some of the complications related to differences in tumor
growth in different animals. [18F](2S,4R)-4-FGln, 2, was
recently reported as a tumor PET imaging agent for
glutaminolysis.8 PET images of [18F](2S,4S)-4-FPGln, 4, and
[18F](2S,4R)-4-FGln, 2, were selected for visualization (Figure
7). As these images demonstrate, the 9L tumors could be
visualized with either of the ligands. High kidney, liver, and
bladder uptake were also observed. Defluorination/bone uptake
was more apparent in the images of [18F](2S,4R)-4-FGln, 2,
compared to those of [18F](2S,4S)-4-FPGln, 4. To assess the in
vivo kinetics, region-of-interest analysis was performed (using
AMIDE software to generate the time-activity curves; Figures 8
and 9). The kinetic curves confirmed that all the tracers
exhibited higher tumor uptake compared to the muscle
(background) regions. [18F](2S,4S)-4-FPGln, 4, showed a
higher tumor-to-muscle ratio than [18F]4-FGln. Both ligands
displayed similar kinetics. Both ligands had rapid tumor uptake
and reached their maximum uptake within the first 20 min.
Tumor uptake for [18F]4-FGln remained rather consistent over
2 h, whereas [18F](2S,4S)-4-FPGln, 4, displayed a faster tumor
washout rate. Also noteworthy, ([18F](2S,4S)-4-FPGln, 4,
showed less defluorination/bone uptake in comparison to
that of [18F]4-FGln. Results of the in vivo PET imaging studies
using the 9L tumor model suggested that the [18F](2S,4S)-4-
FPGln, 4, localized in the 9L tumor as well, if not better, than
[18F](2S,4R)-4-FGln, 2.
Figure 4. In vitro cell uptakes of [18F](2S,4R)-4-FPGln, 3, and
[18F](2S,4S)-4-FPGln, 4. [3H]Gln was used as a standard. All
radiotracers were evaluated in the 9L tumor cell line.
and 4R have comparable tumor cell uptakes. Because of this
observation, we only used the [18F](2S,4S)-4-FPGln, 4, tracer
in further investigations on inhibition of cell uptakes and for the
in vivo biological studies. The tracer, [18F](2S,4S)-4-FPGln, 4,
was incubated at 37 °C for 30 min with different amino acid
transport inhibitors. The results in Figure 5 suggested that the
system A inhibitor, MeAIB (N-methyl-α-aminoisobutyric acid),
had no inhibitory effect on the uptake, indicating that the
system A amino acid transport was not involved in the uptake
of this new tracer. System L inhibitor, BCH (2-amino-
bicylo[2.2.1] heptane-2-carboxylic acid), System ASC inhibitor
L-serine (L-Ser) and System ASC (SLC1A5), N inhibitor, L-
glutamine (L-Gln), exhibited similar concentration dependent
reduction of cell uptake, thus indicating potential involvement
of system L, ASC, and N in the uptake (Figure 5).
Protein Incorporation of [18F](2S,4S)-4-FPGln, 4, into
9L Tumor Cells. One of the important issues to consider when
developing tracers to image glutamine metabolism in tumors is
the protein incorporation of the tracer once inside the cells.
After incubation of [18F](2S,4S)-4-FPGln, 4, and [3H]Gln with
9L tumor cells, the cell lysates were treated with TCA and the
radioactivity in the precipitates and supernatant were counted.
Results showed that the majority of [3H]Gln activity was
associated with the TCA precipitates suggesting that most of
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Figure 5. In vitro cell uptake inhibition studies of [18F](2S,4S)-4-FPGln, 4, conducted in 9L cells using inhibitors: System LAT inhibitor, BCH (2-
amino-bicylo[2.2.1] heptane-2-carboxylic acid); System ASC inhibitor, L-serine (L-ser), System A inhibitor, N-methyl-α-aminoisobutyric acid
(MeAIB); and system N inhibitor, L-glutamine (L-Gln).
a
Table 1. Tissue Distribution of Radioactivity in F344 Rats
Bearing 9L Tumors after Intravenous Injection of
b
[18F](2S,4S)-4-FPGln, 4
organ
blood
30 min
60 min
0.57 0.02
0.31 0.01
0.12 0.01
0.52 0.01
12.4 1.02
3.22 0.42
0.59 0.02
1.72 0.07
0.47 0.15
0.15 0.00
0.53 0.08
0.83 0.04
1.45 0.08
6.91 0.66
0.38 0.02
0.26 0.02
0.11 0.01
0.39 0.02
8.93 0.42
2.24 0.06
0.42 0.03
1.58 0.08
0.37 0.06
0.16 0.01
0.56 0.17
0.60 0.06
1.57 0.17
5.45 0.73
heart
muscle
lung
kidney
pancreas
spleen
liver
skin
brain
bone
9L tumor
tumor/blood
tumor/muscle
Figure 6. Incorporation of [18F](2S,4S)-4-FPGln, 4, and [3H]Gln into
protein in 9L tumor cells was investigated. The comparison of cellular
uptake of [18F](2S,4S)-4-FPGln, 4, and [3H]Gln was performed using
dual-isotope experiments at 0, 5, 30, 60, and 120 min incubation time
periods. Cell lysates were treated with TCA and the radioactivity (both
a
b
Percent dose/gram. Results are expressed as mean SD (n =4).
and rapidly dividing immune cells, and as a regulator of acid−
base balance by producing ammonium in the kidneys. In the
brain, the glutamine−glutamate shunt is a critical pathway to
control the inhibitory and excitatory neuronal signals.
Glutamine transporters play an important role in regulating
mammalian cell functions. There are three known glutamine
transporters, SLC1A5 (ASCT2, Km 20 mM), LAT1 (SLC7A5,
Na+ independent), SNAT (Na+/neutral transporter).16−19 In
the context of tumor growth, SLC1A5 is the most important
glutamine transporter responsible for rapidly growing tumors.
In these tumor cells, the expression of SLC1A5 is up-regulated.
3
18F and H) associated with the TCA precipitates was counted.
DISCUSSION
■
Glutamine is found circulating in the blood as well as stored in
skeletal muscles in high concentrations (0.5−1 mmol/L).
Glutamine plays various critical functions: as an energy source
and a substrate for DNA and protein synthesis, a primary
source of fuel for cells lining the inside of the small intestine
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Figure 7. Representative PET images of 9L tumor bearing rats after intravenous injection of [18F](2S,4S)-4-FPGln, 4, or [18F](2S,4R)-4FGln, 2, into
a F344 rat bearing a 9L tumor. The images of the transverse, coronal, and sagittal views are from a summed 2 h scan. Arrows represent the location
of tumors on the hind leg region of the F344 rat.
Figure 8. Time activity curves of tumor (target) and muscle
Figure 9. Comparison of ratios of tumor/muscle at different time
points (0 to 120 min) for [18F](2S,4S)-4-FPGln, 4, and [18F](2S,4R)-
4-FGln, 2.
(background) uptake in F344 rats bearing 9L tumors: (blue)
[18F](2S,4S)-4-FPGln, 4, and (red) [18F](2S,4R)-4-FGln, 2.
Just as FDG-PET is useful for imaging tumors in which the
glucose transporter is overexpressed, glutamine tracers will
accumulate in these tumors. The tracer reported in this project
appeared to be more sensitive to the inhibition by LAT
inhibitor, BCH, not glutamine (SLC1A5). The relationship
between amino acid transporter expression and tumor growth is
a rapidly expanding research field. Many amino acid derivatives
have been reported for imaging tumor growth based on
different amino acid transporters,20,21 most of which were not
designed to measure glutamine metabolism specifically.
Recently, a detailed study of transport mechanisms of trans-1-
amino-3-fluoro[1-14C]cyclobutanecarboxylic acid (anti-[14C]-
FACBC) has been published. The corresponding
[18F]FACBC is now being tested in humans as a potential
prostate tumor imaging agent.20,22−28 It may be desirable to
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Figure 10. Chemical structures of analogs of glutamine and glutamic acid derivatives: [18F](2S,4R)-4-fluoroglutamine, (2S,4R)-4-FGln, 2, vs
[18F](2S,4R)-4-fluoroglutamic Acid, BAY 85−8050; [18F](2S,4S)-4-(3-fluoropropyl)glutamine, (2S,4S)-4-FPGln, 4, vs [18F](2S,4S)-4-(3-
fluoropropyl)glutamic acid, 18F-FSPG, or BAY 94−9392. These two pairs of probes are structurally very similar, containing a C5 amide vs a C5
carboxylic acid group, but the mechanisms of uptake and retention are dramatically different.
further evaluate the significance of the observation on different
levels of inhibition by LAT vs SLC1A5 subtypes of amino acid
transporters.
amino acid probes are transported into the cancer cells, without
being incorporated into intracellular macromolecules. There are
important differences between these seemingly very close
glutamine analogs, [18F](2S,4R)-4-FGln, 2, and [18F](2S,4S)-4-
FPGln, 4. Further exploration may be needed to clarify the
similarities and differences between these probes. For the
development of effective probes for studying glutamine
metabolism, one should consider more than simple factors,
such as tumor cell uptake and in vivo tumor signal localization.
It may also be necessary to consider the subsequent
intracellular metabolic processes, or the lack thereof. Compared
to the “natural” [11C]Gln, 1, fluorine substituted [18F](2S,4R)-
4-FGln, 2, or [18F](2S,4S)-4-FPGln, 4, may always be
suspected of having a modified intracellular metabolism.
More studies are necessary to characterize these analogs for
studying glutamine metabolism in tumors.
[18F](2S,4R)-4-fluoroglutamic acid, BAY 85−8050 has been
reported as a tumor imaging agent.29 In order to improve the in
vivo stability and to reduce defluorination in vivo, (4S)-4-(3-
[18F]fluoropropyl)-L-glutamate (18F-FSPG, or BAY 94−9392)
was also prepared and tested.30−32 It was found that 18F-FSPG,
a glutamic acid containing a 3-fluoro-propyl substitution group
at the C4 position, showed good tumor uptake and reduced in
vivo defluorination.29 In vivo human studies suggest that 18F-
FSPG is a tracer useful for assessing system xC− (anionic amino
acid) transporter activity in tumors with PET.31,32 Piramal
Biotechnology is now developing the 18F-FSPG for imaging
tumors in which xC− transporters are prominently expressed
and oxidative stress is up-regulated.
The most important difference between [18F](2S,4R)-4-
fluoroglutamic acid (BAY 85−8050) and [18F](2S,4S)-4-(3-
fluoropropyl)glutamic acid (18F-FSPG, or BAY 94−9392) is
that 18F-FSPG, displays a slower defluorination rate in vivo.
Preliminary human studies have demonstrated that the bone
marrow uptake in the vertebral region is relatively low.31,32 The
same scenario may or may not apply to the second pair of
glutamine probes (Figure 10). We have noticed a reduced bone
uptake and good tumor uptake in the rats receiving
[18F](2S,4S)-4-FPGln, 4, as visualized by PET or by a
dissection method. Results from both methods suggest a
reduced bone uptake (of 4) in rats as compared to the uptake
of [18F](2S,4R)-4-FGln, 2.8 Loss of fluoride is a constant
concern for fluoro-alkyl labeled radiopharmaceuticals. Our
observation suggests that defluorination is not an issue for 4.
In summary, a new glutamine analog, [18F](2S,4S)-4-FPGln,
4, has shown tumor specific uptake in vitro and in vivo.
However, the tumor uptake and retention mechanisms may be
significantly different from other glutamine probes, such as
[11C]Gln, 1, and [18F](2S,4R)-4-FGln, 2.
The new (2S,4S)-4-FPGln, 4, reported in this paper, is a
structural analog of glutamine containing a 3-fluoro-propyl
substitution group at the C4 position. The mechanisms of
uptake for [18F](2S,4S)-4-FPGln, 4, are associated with three
main amino acid transporters, SLC1A5 (ASCT2), LAT1
(SLC7A5, Na+ independent), and SNAT (Na+/neutral trans-
porter). Based on the inhibition studies, it appears that LAT
inhibition was the most prominent, suggesting that LAT may
be a preferred transporter for (2S,4S)-4-FPGln, 4. For
[11C]Gln, 1, and (2S,4R)-4-FGln, 2, the most important
amino acid transporter appeared to be SLC1A5 (ASCT2).
The in vitro incubation of [18F](2S,4S)-4-FPGln, 4, with 9L
tumor cells showed a high cell uptake reaching 6% uptake/100
μg of protein at 120 min after incubation. Under the same
incubation conditions, the lysate of the 9L cells showed that a
significant portion of the [18F](2S,4S)-4-FPGln, 4, inside the
cells remained intact as the original chemical species (>90%
showed no metabolic changes). The [3H]-Gln incubated
simultaneously under the same conditions showed substantial
incorporation into macromolecules (>90% activity associated
with the TCA precipitated fraction). Previously, using the same
procedure [11C]Gln, 1, and [18F](2S,4R)-4-FGln, 2, also
displayed very similar incorporations into the macromolecular
fraction as that observed for [3H]-Gln.7,8 The results suggest
that [18F](2S,4S)-4-FPGln, 4, may be more similar to the
neutral LAT preferred amino acid analogs, such as O-(2-
[18F]fluoroethyl)-L-tyrosine (FET).33,34 The uptake mechanism
may be overlapping that of [18F]FACBC.22,35 All of these
AUTHOR INFORMATION
■
Corresponding Author
*Email: kunghf@sunmac.spect.upenn.edu. Phone: 215-662-
3096. Fax: 215-349-5035.
Author Contributions
§Authors Z.W. and Z.Z. contributed equally to this paper.
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Notes
The authors declare no competing financial interest.
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■
The authors thank Dr. Carita Huang for editorial assistance.
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ABBREVIATIONS
■
[11C]Gln, L-5-[11C]glutamine, 1; [18F](2S,4R)-4-FGln, [18F]-
(2S,4R)-4-fluoroglutamine, 2; [18F](2S,4R)-4-FPGln, [18F]-
(2S,4R)-4-(3-fluoropropyl)glutamine, 3; [18F](2S,4S)-4-
FPGln, [18F](2S,4S)-4-(3-fluoropropyl)glutamine, 4; ASC,
alanine-serine-cysteine preferring amino acid transporter
system; ASCT2, system ASC transporter subtype 2; BCH, 2-
amino-bicylo[2.2.1]heptane-2-carboxylic acid; MeAIB, N-meth-
yl-α-aminoisobutyric acid; FACBC, 3-[18F]fluoro-cyclobutyl-1-
carboxylic acid; FC, flash chromatography; FDG, 2-[18F]fluoro-
2-deoxy-D-glucose; FET, O-(2-[18F]fluoroethyl)-L-tyrosine;
[3H]Gln, L-[3,4-3H(N)]-glutamine; HPLC, High-performance
liquid chromatography; HRMS, High-resolution mass spec-
trometry; MeAIB, N-methyl-α-aminoisobutyric acid; TCA,
trichloroacetic acid; TFA, trifluoroacetic acid; PET, positron
emission tomography
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