[68Ga]Ga-DO2A-(OBu-l-tyr)2: Synthesis, 68Ga-radiolabeling and in vitro studies of a novel 68Ga-DO2A-tyrosine conjugate as potential tumor tracer for PET

Article abstract of DOI:10.1016/j.bmcl.2009.05.001

The synthesis, ⁶⁸Ga-labeling and in vitro study of the novel tyrosine chelate derivative [⁶⁸Ga]Ga-1,4,7,10-tetraazacyclododecane-1,7-diacetic acid-4,10-di-(O-butyl)-l-tyrosine ([⁶⁸Ga]Ga-DO₂A-(OBu-l-tyr)₂

Full text of DOI:10.1016/j.bmcl.2009.05.001

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3498–3501
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
[68Ga]Ga-DO2A-(OBu-L-tyr)2: Synthesis, 68Ga-radiolabeling and in vitro studies
of a novel 68Ga-DO2A-tyrosine conjugate as potential tumor tracer for PET
Carsten Burchardt a, , Patrick J. Riss a, , , Frederic Zoller a, Simone Maschauer b, Olaf Prante b,
*
Torsten Kuwert b, Frank Roesch a
a Johannes Gutenberg-University Mainz, Institute of Nuclear Chemistry, Fritz-Strassmann-Weg 2, 55128 Mainz, Germany
b Friedrich-Alexander University, Laboratory of Molecular Imaging, Clinic of Nuclear Medicine, Schwabachanlage 6, 91054 Erlangen, Germany
a r t i c l e i n f o
a b s t r a c t
The synthesis, 68Ga-labeling and in vitro study of the novel tyrosine chelate derivative [68Ga]Ga-1,4,7,10-
Article history:
tetraazacyclododecane-1,7-diacetic acid-4,10-di-(O-butyl)-
potential tracer for imaging tumor metabolism by positron emission tomography (PET) is presented. This
approach combines the biological amino acid transporter targeting properties of -tyrosine with the out-
standing availability of 68GaIII via the 68Ge/68Ga generator. In vitro studies utilizing the F98-glioblastoma
cell line revealed specific uptake of [68Ga]Ga-DO2A-(OBu-
-tyr)2 that was comparable to that of the ref-
erence O-(2-[18F]fluoroethyl)-
-tyrosine (FET). These promising results indicate a high potential of
L L-tyr)2) as a
-tyrosine ([68Ga]Ga-DO2A-(OBu-
Received 3 April 2009
Revised 30 April 2009
Accepted 3 May 2009
Available online 7 May 2009
L
L
Keywords:
F-98 glioblastoma cells
Gallium-68
Macrocyclic ligands
Radiopharmaceuticals
Tyrosine
L
[
68Ga]Ga-DO2A-(OBu-
L
-tyr)2 for molecular imaging of tumor-driven amino acid uptake by PET.
Ó 2009 Elsevier Ltd. All rights reserved.
The influx of amino acids into proliferating cells is mediated by
specific transmembrane transporters, for example, the sodium-
depending systems A and ASC or the sodium-independent system
transport. It is readily available via nucleophilic fluorination.8 The
structural formulas of 1, 2, and 3 are shown in Scheme 1.
It was found that incorporation of the radiolabeled amino acids
into proteins is not necessarily a prerequisite for visualization of
tumors in vivo by PET. Instead, successful targeting of amino acid
transporters appeared to be sufficient for imaging proliferating tu-
mor cells mainly due to enhanced amino acid transport.9 The
acceptance of the above mentioned amino acid derivative 3 by
amino acid carriers indicates a principal structural tolerance
among the variety of amino acid transport systems. In an extension
of this concept, we hypothesize that amino acid transporters may
L
.1 To meet the need of rapidly proliferating tumors for, in particu-
lar, the non-endogenous amino acids, amino acid transport sys-
tems are overexpressed in naturally occurring neoplasms as well
as in certain tumor cell lines.2 This biochemical process can be
visualized in vivo with positron emission tomography (PET), a
quantitative modality of molecular imaging.3 Various positron-
emitter labeled amino acids have already been studied to probe
the uptake and incorporation of amino acids into functional
proteins as well as the amino acid transporter expression.4,5 One
attempt to visualize intracellular protein synthesis rate utilized
(S)-2-amino-4-([11C]methylthio)butanoic acid (1), isotopically
labeled with carbon-11.6 Taking the limited availability of
carbon-11 in a clinical context into account, (S)-2-amino-3-(2-
[
18F]fluoro-4-hydroxyphenyl) propanoic acid (2) was synthesized.
The longer half-life of 18F results in the capability to distribute
the tracer to satellite PET sites. Due to the electrophilic production
route, this tracer did not find systematic application.7
Further improvements involved (S)-3-(4-(2[18F]-fluoroeth-
oxy)phenyl)-2-aminopropanoic acid, FET (3), a compound that is
not incorporated into proteins and thus solely reflects amino acid
-[11C-methyl]-
-tyrosine (2) and O-(2-[18F]fluoroethyl)-
-tyrosine
* Corresponding author. Tel.: +49 6131 3925701.
E-mail address: riss@uni-mainz.de (P.J. Riss).
These authors have contributed equally to this work.
Scheme 1. Structural formulas of the radiolabeled amino acids
L
methionine (1), 2-[18F]fluoro-
(3).
L
L
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.001

C. Burchardt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3498–3501
3499
tolerate a conjugation of even more complex structural compo-
nents. This may in particular create the option to introduce
chelators to the amino acids, for example, for radiolabeling with
metallic radionuclides such as 68GaIII, a promising positron emitter
derived from the commercially available 68Ge/68Ga-radionuclide
generator.10
68Ga exhibits a high positron abundance of 89% and a half-life
(
68Ga, t1/2 = 1.13 h) well suited for PET. The relation between moth-
ers (68Ge, t1/2 = 270.8 d) and daughters half-life provides the possi-
bility to elute 68GaIII as 68GaCl3 from the generator multiple times a
day. 68Ge/68Ga-radionuclide generators can be used for about one
year. Due to this fact this radionuclide generator system provides
increased availability and significant cost reduction compared to
the cyclotron-produced nuclides.
However, the challenge in preparing (radio)metal-containing
amino acids with retained biological activity is to develop an
appropriate synthetic strategy. In the case of many metals, cova-
lent binding to amino acid structures is not an option. The intro-
duction of bifunctional chelate ligands may represent an
alternative approach.11
L-Tyrosine appears to be a promising lead
for the introduction of a chelating moiety. This is due to the free
aromatic hydroxyl group, tolerating chemical modification as
shown for FET.
In this study, we report the feasibility of generating a 68GaIII-la-
beled tyrosine derivative that reveals specific uptake in glioblas-
toma cells in vitro. Various different approaches to couple
targeting molecules to a macrocyclic ligand have been reported
so far.12 Herein the utility of the two ring-nitrogen atoms of
1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A) to attach
two tyrosine moieties via an alkyl spacer is described for the first
time. With this concept we intend to increase the intramolecular
amino acid concentration of the compounds, while maintaining
the overall molecular weight in a reasonable range.
The synthesis route to the precursor 9 is shown in Scheme 2. A
tert-butyl-protected DO2A (7) was synthesized in three steps start-
ing from cyclen with an overall yield of 68%.13 Two
L-tyrosine moi-
eties were introduced to form a bivalent DO2A-tyrosine conjugate.
To compensate potential affinity-decreasing side effects of a bulky
and charged GaIII-chelate, we inserted a butyl spacer unit between
the hydroxyl group of
cyclic chelator.
L-tyrosine and a ring-nitrogen of the macro-
The methylester, tert-butylester and BOC protective groups of 8
were subsequently cleaved employing standard reaction condi-
tions. Purification of the resulting labeling precursor
9 was
performed by HPLC, and the TFA-salts were removed using an
ion-exchange resin.14
Scheme 2. Synthesis route to the labeling precursor 9. Reagents and conditions: (a)
1-bromo-4-chlorobutane, K2CO3, acetone, reflux, 91% yield, (b) NaI, acetone, 94%, (c)
N,N-diisopropylethylamine, MeCN, 60 °C, 42% yield, (d) 1 M NaOH/dioxane, (e) TFA,
60% yield (over two steps).
68Ga-labeling of the precursor 9 was carried out in aqueous solu-
tion at 90 °C with 50 l
l 68Ge/68Ga-generator eluate, containing puri-
fied no carrier added 68GaCl3.15 The labeled product (Scheme 3) was
obtained in 94 2% yield after 5 min.
The time dependency of labeling yields for 10 is given in Figure 1.
The radiochemical purity after solid phase extraction was greater
than 98% as analyzed by radio-TLC and radio-HPLC.
The radiolabeled product was formulated in phosphate-buf-
fered saline (PBS) buffer (pH 7.4), and sterile-filtered prior to appli-
cation. Stability of the 68Ga-labeled complex 10 was proven in a
diethylentriamine-penta-acetic acid (DTPA)- and an apo-transfer-
rin challenge experiment at 37 °C for 2 h. The results indicate ade-
quate stability with about 99% of 68Ga-labeled DO2A-(OBu-
remaining intact within this period of time (Fig. 2).
L-tyr)2
In vitro studies for the determination of specific uptake of the
novel tracer 10 were performed utilizing a F98 rat glioblastoma cell
line.
This in vitro model has successfully been used previously in
various studies which aimed at characterizing amino acid transport
of tracers.17 The time-dependency of uptake was followed over
Scheme 3. Structural formula of [68Ga]Ga-DO2A-(OBu- -tyr)2 (10).16
L

3500
C. Burchardt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3498–3501
Figure 4. Comparison of the specific binding of [18F]FET (3) (circles, n = 3) and
68Ga]Ga-DO2A-(OBu-
-tyr)2 (squares n = 6) to F98 glioblastoma cells at 4 °C. Tracer
uptake values were expressed as mean values related to the protein mass (%/
mg) one standard deviation.
Figure 1. Radiochemical yields (RCY) of 10 by labeling of DO2A-(OBu-L-tyr)2 (9)
[
L
with 68Ga at three different temperatures (90 °C (squares), 70 °C (circles), 50 °C
(triangles)) in aqueous solution. Labeling yields are given as mean values
(%) standard deviation.
parison. The 68Ga-labeled derivative 10 showed a continuously
increasing cellular uptake over 90 min. The total uptake of 10
was reduced to the level of non-specific binding in the presence
of a mixture of L-tryptophan (Trp), L-serine (Ser) and 2-aminobicy-
clo-(2,2,1)-heptane-2-carboxylic acid (BCH).
These results indicate specific uptake of 10 by F98 cells, pre-
sumably due to the interaction with an amino acid transporter.
In order to allow a reasonable comparison between uptake val-
ues of the novel compound 10 and [18F]-3 as a reference, the tracer
uptake was normalized to the protein concentration of each sam-
ple determined by the method of Bradford.17 As shown in Figure
4, (S)-3-(4-(2[18F]-fluoroethoxy)phenyl)-2-aminopropanoic acid,
FET (3) and 10 showed specific uptake to F98 rat glioblastoma cells
within a similar range.
In conclusion, the presented data illustrate a novel chemical ap-
proach to synthesize a metal-containing amino acid derivative
with potential for molecular imaging. This study stimulates further
work capitalizing on the potential of the positron emitter 68Ga or
other metals for developing specific probes of amino acid metabo-
lism in tumors. Work is in progress to use 10 for in vivo imaging of
F98 glioblastoma-bearing rats by small-animal PET.
Figure 2. Challenge experiment of [68Ga]Ga-DO2A-(OBu-
in two concentrations (1 mmol, 750-fold excess; squares;
circles) and 5 ml transferrin solution (1 mmol, 375-fold excess, triangles) at 37 °C
over 120 min (n = 3). Stability values were expressed as mean values of intact tracer
(%) one standard deviation.
L
-tyr)2 with 10 ml of DTPA
1
lmol, 0.75 equiv,
Acknowledgments
The authors want to thank the ‘Fonds der chemischen Industrie’
for the generous donation of chemicals and the COST D38 action
for networking all over Europe.
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