A simple route to [11C]N-Me labeling of aminosuberic acid for proof of feasibility imaging of the xC- transporter

Article abstract of DOI:10.1016/j.bmcl.2014.10.004

Oxidative stress has been implicated in a variety of conditions, including cancer, heart failure, diabetes, neurodegeneration and other diseases. A potential biomarker for oxidative stress is the cystine/glutamate transporter, system x_C^-. l-Aminosuberic acid (l-ASu) has been identified as a system x_C^- substrate. Here we report a facile method for [¹¹C]N-Me labeling of l-ASu, automation of the radiochemical process, and preliminary PET imaging with EL4 tumor bearing mice. The results demonstrate uptake in the tumor above background, warranting further studies on the use of radiolabeled analogs of l-ASu as a PET imaging agent for system x_C^-.

Full text of DOI:10.1016/j.bmcl.2014.10.004

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5512–5515
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A simple route to [11C]N-Me labeling of aminosuberic acid for proof
of feasibility imaging of the xÀC transporter
Hua Yang a, Qing Miao a, Bruce F. Johnson b, Michael J. Rishel b, Vesna Sossi c, Katherine Dinelle c,
François Bénard. d,e, Donald T. Yapp f,g, Jack M. Webster b, Paul Schaffer a,e,
a Nuclear Medicine Division, TRIUMF, Vancouver V6T 2A3, Canada
⇑
b Diagnostics and Biomedical Technologies, GE Global Research, Niskayuna, NY 12309, USA
c Department of Physics and Astronomy, University of British Columbia, Vancouver V6T 1Z1, Canada
d Centre of Excellence for Functional Cancer Imaging, British Columbia Cancer Agency, Vancouver V5Z 4E6, Canada
e Department of Radiology, Faculty of Medicine, University of British Columbia, Vancouver V5C 1M9, Canada
f Experimental Therapeutics, British Columbia Cancer Agency, Vancouver V5Z 4E6, Canada
g Division of Pharmaceutics and Biopharmaceutics, Faculty of Pharmacy, University of British Columbia, Vancouver V6T 1Z3, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 July 2014
Revised 27 September 2014
Accepted 1 October 2014
Available online 13 October 2014
Oxidative stress has been implicated in a variety of conditions, including cancer, heart failure, diabetes,
neurodegeneration and other diseases. A potential biomarker for oxidative stress is the cystine/glutamate
transporter, system xÀC .
L
-Aminosuberic acid (
report a facile method for [11C]N-Me labeling of
liminary PET imaging with EL4 tumor bearing mice. The results demonstrate uptake in the tumor above
L
-ASu) has been identified as a system xÀC substrate. Here we
L
-ASu, automation of the radiochemical process, and pre-
background, warranting further studies on the use of radiolabeled analogs of
L-ASu as a PET imaging
Keywords:
Radiolabelling
C-11
agent for system xÀC .
Ó 2014 Elsevier Ltd. All rights reserved.
Oxidative stress
Cystine transporter
N-methylation
Amino acids play an important role in many biological pro-
cesses, serving as the building blocks for protein synthesis and as
substrates for important intermediary metabolic processes and cell
signaling pathways.1 Reports on the effects of intra- and extracel-
lular availability of many amino acids are growing, suggesting that
amino acids are critical nutrients that can regulate many aspects of
cellular physiology by modulating gene expression and signal
transduction pathways.1–3 For this reason, radiolabeled amino
acids have become increasingly attractive tools in the search for
novel molecular imaging probes3,4 as alternatives to complement
18FDG imaging. Many 11C- and 18F-labeled amino acids have been
reported,5–10 providing varying degrees of imaging information
based on the rate and mechanism of transport for each tracer
toward their intended amino acid transporter target.
uptake,7,8,10,12 however, we are interested in exploiting system
xÀC , a cystine/glutamate antiporter with a very narrow natural
substrate binding profile,13–18 yet sufficient promiscuity and
upregulation in the disease state to be a potentially useful target
for PET.19–21 The upregulation mechanism of system xÀC is illus-
trated in Figure 1a. Of the standard amino acids, only L-glutamate
(L-Glu) and L
-cystine are substrates for system xÀC .22 In strategizing
for both 11C- and 18F-labeled derivatives suitable for xÀC imaging,
we examined cystine and glutamate, both of which possess limita-
tions as precursors. Cystine has a potentially labile disulfide bond,
while our concern with the use of L-glutamate derivatives lies in
the presence of alternative glutamate receptor and transporter
mechanisms in addition to system xÀC (e.g., EAATs); potentially
complicating in vivo pharmacokinetics23 and eventual clinical
The transfer of amino acids across the plasma membrane is
mediated by transporters for which there are over 20 unique sys-
tems identified with multiple related isoforms.1,11 Many existing
amino acid based tracers target the Na dependent neutral amino
interpretation. Our hypothesis was that L-aminosuberic acid
(L-ASu) would exhibit some level of binding affinity toward system
xÀC while avoiding the structural shortcomings of cystine and the
perceived specificity shortcomings associated with glutamate
(Fig. 1b).
acid transporters system
L
and system ASC for tumor
Previously, our group has studied N-labeled analogs of cystine as
system xÀC substrates but obtained mixed results in our in vitro and
in vivo tumor uptake studies. Initially, we sought to label cystine
⇑
Corresponding author. Tel.: +1 604 222 7696; fax: +1 604 222 1074.
E-mail address: pschaffer@triumf.ca (P. Schaffer).
http://dx.doi.org/10.1016/j.bmcl.2014.10.004
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

H. Yang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5512–5515
5513
Cys-S-S-Cys
(a)
Glu
-
xc
Cys-SH
G-S-S-Cys
Cys-S-S-Cys
GSH
Glu
Cytosol
GSSG GSH
Cys-SH
Glu
Nrf2
ROS
ROS
ROS
Glu-Cys
Nrf2
Active ARE
KEAP1[-S-R]
GSH
Nucleus
GR
Detoxified Products
265 Proteasome
GSSG
(b)
O
O
O
O
NH2
OH
S
OH
HO
OH
HO
HO
S
NH2
NH2
O
NH2
O
L-ASu
L-Glu
L-Cysꢀne
Figure 1. (a) Mechanism of system xCÀ upregulation during oxidative stress. In cytosol, cystine (Cys-S-S-Cys) uptaken by system xÀC is reduced to cysteine (Cys-SH) which is
the rate limiting precursor for glutathione (GSH) synthesis. GSH eliminates reactive oxygen species (ROS). During oxidative stress, High ROS level triggers the modification of
a ubiquitin ligase KEAP1, which can no longer facilitate the degradation of Nrf2. Nrf2 is a transcript factor that can active the expression of antioxidant response element
(ARE) promoters, including system xÀC . (b) Structures of
L-cystine, L-Glu and L-ASu.
O
with a fluorophore to establish feasibility and understand the
impact of a sterically-demanding group on the pharmacokinetic
profile of cystine. Mono-N-bodipy labeled cystine demonstrated
high in vitro labeling of apoptotic cells,24 however, further study
using an [18F]fluorobenzaldehyde-labeled aminoxy derivative of
cystine suffered from poor imaging contrast in A549 tumor bearing
mice with high blood uptake.24 The contradicting results obtained
with bodipy- and fluorobenzaldehyde-modified tracers forced us
to revise our hypothesis regarding large prosthetic groups for label-
ing cystine analogs. Instead, we sought to minimize the size of the
prosthetic group, prompting us to examine [11C]Me- as the pros-
thetic group of choice for in vivo proof of feasibility studies. There-
O
11CH3I,
R
O
R
O
O
R
O
R
Ag2O or Cs2CO3
NH
O
NH2
O
H311C
3
4
R=PMB
R=Me
O
HO
O
OH-
OH
+
OH
11CH3OH
+
HO
NH
O
NH2
O
H311C
[
11C]N-Me
L-ASu
Scheme 2. Radiosynthesis of [11C]N-Me
L
-ASu from L-ASu di-ester.
fore, we designed [11C]N-Me
cystine transporter. In this report, we disclose the radiosynthesis
of [11C]N-Me
-ASu and its in vivo performance in EL4 xenograft-
L-ASu as the tracer targeting the
1 and 2). Figure 2a illustrates that there are three peaks on HPLC
of which the earliest peak (5.2 min, 17 4% n = 3) corresponded
L
to [11C]N-Me
L-ASu while the later eluting peaks were assigned
tumor bearing mice. PET imaging of this tracer provided lower
background compared to large 18F prosthetic group labeled cystine
analogs, demonstrating that a smaller labeling group may give
more favorable biodistribution in vivo.
to 1 and 2. The 11C methyl esters were cleanly hydrolyzed in the
presence of NaOH at 100 °C for 5 min, as was evident by the corre-
sponding shift in HPLC retention times following saponification
(Fig. 2b).
We examined two methods to label
L
-ASu at the amino position.
-ASu
-ASu ana-
The first is to add
[
11C]-MeI directly to unprotected
L
Following hydrolysis, the reaction mixture consisted of the
desired product along with the precursor (L-ASu), cesium carbon-
(Scheme 1). The alternative is to use carboxyl-protected
log as the precursor (Scheme 2).
L
ate, sodium hydroxide, [11C]methanol and DMSO. To afford purifi-
cation of the desired product without HPLC, we recognized that
Direct preparation of [11C]N-Me
L
-ASu was attempted by treat-
-ASu with [11C]MeI and cesium carbonate in DMSO
(Scheme 1). As expected, this reaction resulted in the production
of three 11C labeled compounds: the desired N-methyl
-ASu and
N-Me
L-ASu would maintain a formal negative charge under
ment of
L
neutral to basic conditions, allowing us to separate it from the
remaining reaction components using a strongly anionic exchange
resin eluted by increasing concentrations of sodium. Using a
L
the corresponding
a- and
x
-methyl esters of
L-ASu (Compounds
O
O
11CH3I, Cs2CO3
OH
OH
HO
HO
DMSO, 100oC, 5 min
NH
[
O
NH2
O
H311C
Step 1
11C]N-Me
L-ASu
L
-ASu
O
O
H311C
O
OH
O
+
+
HO
O
11CH3
+
NH2
O
NH2
O
2
1
O
NaOH, H2O
100oC, 5 min
Step 2
OH
OH
HO
H311C
+
11CH3OH
HO
NH
O
NH2
O
[
11C]N-Me
L
-ASu
L
-ASu
Scheme 1. Direct labeling of
L
-ASu with [11C]MeI.

5514
H. Yang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5512–5515
studies using dimethyl ester (4) as precursor showed no improve-
O
O
H311C
OH
OH
ment. Better results were obtained with Ag2O/DMF (17 15% HPLC
RCY, n = 3), however it is difficult to use Ag2O powder in an auto-
mated system (see below). Given the results of labeling both
HO
O
O
NH2
O
NH
H311C
1
11
N-Me
C]
L
-ASu
[
O
Solvent
front
O
11CH3
OPMB- and OMe-protected
cially available, unprotected
L
-ASu, we chose to use the commer-
HO
NH2
O
2
L
-ASu as the precursor for PET stud-
l
ies, sacrificing RCY in the interest of safety and reproducibility
(a) Step 1, γ trace
(b) Step 2, γ trace
provided by using an automated synthesis module.
Given the short half-life of 11C and the relatively long overall
synthesis times and large amounts of activity used, we developed
an automated synthesis on a GE TRACERLab FX CPro module to
enable tracer production for small animal imaging studies.
[
11C]CH4 was produced from a TR19 cyclotron and transferred to
the module before trapping on a liquid nitrogen cooled Poropak
Figure 2. HPLC chromatograph
1, C11-methylation. (b) Step 2, hydrolysis.
c traces of radiolabeling reaction mixture. (a) Step
Q column. [11C]CH4 was converted to [11C]MeI and transferred to
the reaction vial containing L-ASu, Cs2CO3 and DMSO. After the
reaction and hydrolysis, the onboard TracerLab HPLC system was
bypassed and the reaction mixture was transferred to the anion
exchange column and the product was eluted as discussed above
(see time list in Supplementary material). Using this module, the
Dowex 1X8 resin (trialkyl ammonium chloride resin), [11C]N-Me
L-
ASu isolation was most effectively accomplished by first washing
the column with 3 mL deionized water followed by 3 mL 0.1 Â PBS
to remove most of the radioactive impurities, cesium carbonate,
conversion from
10 1 min after EOB (n = 6, decay corrected). The RCY from
11C]MeI to final sterilized 11C]N-Me
-ASu is 5.1 2.0%,
30 3 min after EOB (n = 6, decay corrected). Specific activity was
calculated as the activity of [11C]N-Me
-ASu over the amount of
-ASu: 3.7 1.9 Ci/mmol.
Performing in vitro studies with C-11 is challenging due to the
[ [
11C]methane to 11C]MeI was 77 4% in
DMSO and part of OHÀ and
L
-ASu. The product was eluted from
the resin in 1.5 mL of 1Â PBS buffer with high radiochemical purity
(>98%, Fig. 3a). Separately, we determined that -ASu did not epi-
[
[
L
L
merize under the strongly basic conditions used for ester hydroly-
sis (see Supplementary material).
L
L
l
The pH of the product-containing fraction was adjusted to 6–8
by the addition of a small amount of 1 M HCl, typically ꢀ20
RCY from [11C]MeI to purified [11C]N-Me
-ASu was 10.3 5.4%, in
46 4 min after EOB (decay corrected, n = 3). The identity of the
product [11C]N-Me
-ASu was established by correlative HPLC
against authentic non-radioactive standard (Fig. 3). The effective
specific activity was calculated to be 1.7 Ci/mmol based on the
presence of excess
-ASu. In order to separate the 11C-labeled prod-
uct from -ASu, it was necessary to start with <1 mg of the starting
material in order to obtain [11C]N-Me
-ASu with a total concentra-
tion of contaminating -ASu less than the endogenous blood cys-
lmol.
short t1/2. We attempted blocking studies with sulfasalazine, a
known system xÀC inhibitor using EL4 cells. At 20 min, the cell
uptake was reduced by 91 41% in the presence of sulfasalazine
(n = 3). Despite the challenge with the assay, the blocking effect
of sulfasalazine was evident, prompting us to pursue the analysis
L
L
of [11C]N-Me
L-ASu in vivo.
l
L
A preliminary PET imaging study was carried out on Rag2M
mice bearing EL4 xenograft tumors. A representative image (aver-
age frames from 52.5 to 67.5 min) is shown below (Fig. 4). Tracer
uptake in the kidneys, bladder, heart and tumor is evident. There
is also uptake in the nasal cavity, brain, liver, blood and muscle,
however all are low relative to the tumor. The centre of the tumor
has low activity likely due to necrosis.
L
L
L
tine concentration (<100 lM). This is to avoid concomitant
inhibition of system xÀC by unlabeled ASu during subsequent
studies.25,17
lPET
Despite the convenience of using a commercially available,
Region of Interest analysis (%ID/g) shows tracer uptake for
heart, kidney, muscle and tumor from 0 to 60 min in Figure 5.
There was a rapid tracer uptake in the first 5 min post injection.
The heart uptake peaked at 1.5 min, probably due to the blood dis-
tribution of the tracer and then dropped sharply between 1.5 and
5 min. The kidney uptake reached a maximum at 4.5 min followed
by a rapid reduction between 5 and 10 min. Meanwhile, the tracer
unprotected precursor such as
L
-ASu, much of the 11CH3I activity
was not incorporated in the final product. This is due to the simul-
taneous reaction with the unprotected carboxylates. We decided to
explore an alternative approach using a carboxyl-protected
analog as our precursor for 11C incorporation (Scheme 2). Starting
from di-p-methoxybenzyl (PMB) -ASu ester (3), and using identi-
L-ASu
L
cal conditions as for the direct labeling approach gave >5 radioac-
tive impurities after reaction with 11CH3I. After deprotection,
>98% activity was impurity (see Supplementary material). Further
(a) [11C]N-Me L-ASu,
γ trace
tumor
tumor
tumor
(b) Co-injection with
N-Me L-ASu, ELSD
(c) N-Me L-ASu, ELSD
Sagittal
Transver
Corona
Figure 3. HPLC chromatograph of resin purified [11C]N-Me
co-injection of cold N-Me -ASu (b, evaporation light scattering detector (ELSD)
trace) compared to the cold standard alone (c, ELSD trace).
L-ASu (a, c trace) with
L
Figure 4. Images obtained from the microPET scan of an EL4 tumor xenographed
mouse (408 Ci injected, image summed over 52.5–67.5 min).
l

H. Yang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5512–5515
5515
L
-ASu to image xÀC activity in vivo. Our method starts from a com-
mercially available precursor and was automated to facilitate rapid
manipulation of large amounts of starting activity as required
when working with a short half-life isotope. While not explored
in this work, increased yields may be possible through the use of
[
11C]methyl triflate instead of [11C]methyl iodide, or by imple-
menting the loop method to increase the reaction surface area.26
In vivo PET imaging demonstrated that the small prosthetic
amine labeling methodology improved the biodistribution profile
compared to more sterically demanding labeling groups explored
in our previous work. The tumor-to-background ratio increased
over time, but the optimal imaging contrast was limited by the
half-life of 11C.
Acknowledgments
Figure 5. ROI analysis of [11C]N-Me
L-ASu uptake in various organs.
The authors thank the TR13 staff and radiopharmaceutical pro-
duction radiochemists at TRIUMF, as well as the TR19 operators,
radiochemists and animal facility staff at BCCA for their help.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.10.004.
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uptake in tumor reached a plateau at 3.5 min and maintained this
level indicating very slow clearance. After 40 min, the uptake in
tumor is the second highest, second only to kidney. The tumor/
muscle ratio started at 1.5 at 0.5 min, increased to 2.5 at 30 min
and quickly reached above 4 at 55 min (Fig. 6). The tumor/heart
ratio increased rapidly from 0 to 0.5 in the first 5 min and contin-
ued to increase to above 1 at 30 min. Due to low activity, data from
after 60 min were not analyzed. These ratios were favorable when
compared to earlier studies in which an [18F]fluorobenzaldehyde
labeled aminoxy derivative of cystine showed high blood retention
in a 4 h in vivo biodistribution study.24 Using N-Me labeled
L-ASu
gives much better imaging contrast. However, it is clear that the
tumor imaging quality is not maximized within this time frame
because while the tumor/heart and tumor/muscle ratio were still
increasing, the activity level dropped rapidly due to the short
half-life of 11C, preventing the contrast to reach maximal. Given
these results, we felt, and have since established that an 18F labeled
ASu derivative could potentially provide better contrast and a
longer time for image acquisition.18
In summary, a facile method for 11C amino labeling of
L-ASu has
been developed, providing sufficient tracer quantities to enable
26. Wilson, A. A.; Garcia, A.; Jin, L.; Houle, S. Nucl. Med. Biol. 2000, 27, 529.
preclinical data analysis on the feasibility of using [11C]N-Me