Synthesis of 18F-radiolabeled diphenyl gallium dithiosemicarbazone using a novel halogen exchange method and in vivo biodistribution

Article abstract of DOI:10.1002/jlcr.3746

¹⁸F-radiolabeled diphenyl gallium thiosemicarbazone was prepared by [¹⁸F] fluoride exchange of a nitrato anion under mild conditions. The diphenyl gallium thiosemicarbazone chloride is easily prepared in gram quantities and can be us

Full text of DOI:10.1002/jlcr.3746

Venkatachalam Taracad (Orcid ID: 0000-0001-9747-1105)
Bhalla Rajiv (Orcid ID: 0000-0003-1421-3667)
Lawrie Ken (Orcid ID: 0000-0002-8061-3696)
Synthesis of 18F-radiolabeled diphenyl gallium dithiosemicarbazone using a
novel halogen exchange method and in vivo biodistribution.#
T.K. Venkatachalam*1, D.H.R. Stimson1, R. Bhalla1, K. Mardon1, Paul.V. Bernhardt2, D.C.
Reutens1
1Centre for Advanced Imaging and 2School of Chemistry and Molecular Biosciences, The
University of Queensland, Brisbane 4072.
*Corresponding Author: t.venkatachalam@uq.edu.au
Abstract:
18F-radiolabeled diphenyl gallium thiosemicarbazone was prepared by [18F] fluoride exchange
of a nitrato anion under mild conditions. The diphenyl gallium thiosemicarbazone chloride is
easily prepared in gram quantities and can be used at room temperature in the presence of
oxygen. The corresponding nitrate complex is prepared using silver nitrate in methanol solvent
and can be stored under nitrogen for weeks before radiolabeling. The biodistribution of this
new tracer was studied in mice using Positron Emission Tomography (PET).
Keywords: Synthesis, gallium, dithiosemicarbazone, radiochemistry, biodistribution.
___________________________________________________________________________
# This paper is dedicated to Prof. Leone Spiccia (Monash University, Australia) a friend, scholar
and mentor for his dedicated and tireless contribution to science.
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jlcr.3746
This article is protected by copyright. All rights reserved.

Introduction:
Thiosemicarbazones and semicarbazones can coordinate with many metal ions because of the
presence of several donor atoms such as nitrogen, oxygen and sulphur in their structural core.
The preparation and characterization of diversely substituted thiosemicarbazones and
semicarbazones has been explored since the 1950s 1 and extensive studies of coordination
2
3
compounds with metal ions were undertaken in the following decade . Lobana et al. have
compiled a detailed review of the bonding and structural characteristics of thiosemicarbazones
3-11
and reported strategies to prepare new derivatives. Several workers in the past
have
explored the possibility of introducing various metal ions in the core structure of
thiosemicarbazone and extensively studied their structure and biological activity.
12-24
Dithiosemicarbazones readily coordinate 3d-transition metal ions
and have been used to
8,31-33
chelate positron emitting isotopes such as copper-64 and gallium-68 25-30. Pascu et al.
synthesized a variety of 68Ga-complexes of dithiosemicarbazone as potential imaging agents.
31
Pascu et al
also used a transmetallation strategy to radiolabel zinc complexes of
dithiosemicarbazones with 64Cu. This was accomplished with an aim to use the probe in
hypoxia related imaging modality. Furthermore, the authors indicated that their strategy could
18
serve as target specific site in vivo imaging studies. Since F is more widely available than
64Cu and has a longer half-life than 68Ga, we sought a facile method to introduce 18F in the core
structure of gallium complexes of dithiosemicarbazones and explored whether Ga-18F
dithiosemicarbazone complexes can be potentially used for in vivo imaging. These compounds
form a square pyramidal structure, raising the possibility of introducing positron emitting
isotopes easily using a halogen exchange strategy. Although this method of introducing
fluorine-18 has been explored by McBride et al. 34-39 and Bhalla et al. 40-43 using different metal
chelate moieties, the complexes generated by these groups are all hydrophilic. In contrast,
gallium-18F complexes of dithiosemicarbazones will be both lipophilic and neutral. We
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44
recently reported
a process for introducing [18F]fluoride into the core structure of
dithiosemicarbazones using a halogen exchange method under mild conditions. Here, we
describe semi-automated synthesis of this radiotracer, which includes the purification of the
tracer and report its biodistribution in mice.
Experimental:
All chemicals were obtained from Sigma Aldrich and used without further purification.
Anhydrous methanol and dimethyl sulfoxide also obtained from Sigma Aldrich were stored
under nitrogen and transferred into flasks for synthesis under nitrogen atmosphere to eliminate
any moisture. Sodium methoxide was also kept and transferred under nitrogen atmosphere.
Synthesis of the gallium chloro dithiosemicarbazone, its characterization along and initial
radiolabeling studies have been reported earlier 44.
Preparation of gallium nitrate dithiosemicarbazone: In a scintillation vial was placed 20 mg
of the red complex of gallium chloro dithiosemicarbazone, into which was syringed in 10 mL
of anhydrous methanol under nitrogen atmosphere. The contents were allowed to stir for 10
min; the complex did not dissolve at this stage. To the suspension was added carefully 8 mg of
solid silver nitrate and the contents of the flask manually shaken for 3 min and stirred
vigorously for another 10 min, when the solution turned a reddish yellow color and a white
precipitate of silver chloride was seen (Note: Manual shaking was found to be essential to
initiate the reaction due to heterogeneity of the reaction medium). The contents of the
scintillation vial were then transferred into a 20 mL centrifuge tube under nitrogen and
centrifuged at 3000 rpm for 5 min. A 0.5 mL aliquot of the orange/yellow supernatant was
transferred into a 10 mL glass vial and the solvent evaporated under nitrogen to obtain an
orange/yellow residue. After thorough drying of the residue, the vial was crimp-capped with a
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butyl rubber closure. This intermediate reactive nitrate complex could be stored under these
conditions for several days without adversely affecting radiolabeling.
Radiolabeling of the precursor: A reproducible method was achieved after several trials and
the detailed steps are described below.
Production of fluorine-18: No-carrier-added fluorine-18 was manufactured via the
18O(p,n)18F nuclear reaction using a Cyclone® 18 Twin (IBA, Belgium) dual ion source
cyclotron. Approximately 0.7 mL of pure water enriched to >98% [18O] H2O was irradiated
with 18 MeV protons at a beam current of 14 µA for up to 30 min producing up to 30 GBq of
fluorine-18. The aqueous [18F] F- was transferred from the cyclotron target to a 10 mL glass
receiving vial located in a hot-cell via 1.6 mm diameter (OD) polypropylene tubing
approximately 20 m in length under helium pressure. The receiving vial was measured for
radioactive content in a dose calibrator housed within the hot-cell before delivery into a lead
pot. The lead pot was then manually transferred to a fume cabinet where aliquots of the
aqueous [18F]F- were taken for radiolabeling.
Analytical HPLC conditions for determination of radiolabeled compound: HPLC analysis
was performed using a Shimadzu model LC 20AD instrument with a diode array detector, a
quaternary pump, thermostated column compartment, and a radio detector (Bioscan Flow
Count with a pin-diode detector) in conjunction with a LCsolutions software computer operated
system. The analytical column used was a ZORBAX Eclipse Plus C18 (Agilent) of dimensions
4.6 X 150 mm, 5 µm. The temperature of the column was maintained at 30°C during the
analysis. The eluent used was a mixture of 0.1% TFA in water (A) and Acetonitrile (B) under
gradient conditions of 10 - 90% B over 30 min. The flow rate employed was 1 mL/min and the
injection volume were 20 µL. Under these experimental conditions, the radiolabeled compound
eluted at 15.6 min.
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Preparation of [18F] fluoride /K222/K2CO3: All manual operations were performed behind lead
blocks arranged within protective biological safety cabinets and employing Personal Protective
Equipment (PPE). To dry the [18F] F-, 1.9 mg (5 µmol) of Kryptofix® 222 (K222) was first
dissolved in 0.5 mL of dry acetonitrile and transferred into a 10 mL glass Wheaton vial. To this
was added 25 µL of 0.1 M K2CO3 (2.5 µmol) followed by 100 L of the [18O] H2O containing
cyclotron-produced [18F] F- (typically 200-300 MBq). The vial was septum capped, placed in
a dry block heater maintained at 100°C and heated under nitrogen making sure that the
azeotropic evaporation of the solvent took place smoothly without bumping or spurting.
Evaporation was continued for approximately 20 min resulting into a dry solid mixture of [18F]
F-/K222/K2CO3.
Radiolabeling protocol: The vial containing [18F] F-/K222/K2CO3 was removed from the heater
and set aside while the heating block was cooled to 45°C using compressed air flow. The
gallium nitrate dithiosemicarbazone was dissolved in anhydrous dimethyl sulfoxide (500 L)
resulting in a light yellow-orange solution. The solution was transferred, using a dry syringe
previously flushed with nitrogen, to the vial containing [18F] F-/K222/K2CO3 and a red-orange
colored solution resulted. The vial was then placed in the heater block at 45ºC and allowed to
react for 3 min. The crude reaction mixture was further purified and reformulated using the
Synthra automated module.
Purification of labeled crude reaction mixture: Purification used a combination of semi-
preparative HPLC and Solid Phase Extraction (SPE) using a pre-programmed automated
sequence in a Synthra radiosynthesis module (Synthra GmbH, Hamburg, Germany) housed
within a hot-cell (Tema Sinergie, Faenza, Italy). To separate the radiolabeled compound from
the starting material, HPLC was performed using a ZORBAX Eclipse XDB-C18 250 x 10 mm
semi-preparative column (Agilent, Santa Clara, Ca) and a mixture of 1% trifluoroacetic acid in
water/acetonitrile (65:35) as the eluent. Chromatographic separation was achieved isocratically
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at a flow rate of 4 mL/min. Radiation and UV detection at 254 nm were used to identify the
product. The SPE cartridge used was a Waters C-18 Plus Sep-Pak (Waters Corporation,
Milford, Ma) preconditioned with 10 mL of ethanol and 10 mL of water followed by drying
with air.
The crude product mixture was syringed out using a disposable syringe and diluted to 1 mL in
water (approximately 50% dilution). The syringe was connected manually to position 3 of the
syringe valve of the Synthra module (see Figure 1). In the automated sequence program, the
solution was injected into the 3 mL HPLC loop and peak collection at approximately 14 min
was achieved using the Synthra module’s peak-cut feature. The product was then transferred
into a vial containing 30 mL of water and the contents stirred for 5 min before being transferred
onto the preconditioned SPE cartridge via valve 35 with the solution directed to waste (via
valve 36). The SPE cartridge was then washed with 10 mL of water from vial C3. The product
was then eluted from the SPE cartridge using 1.0 mL of ethanol supplied from vial C2 and
directed to the product vial via valve 36. The product vial was housed in a lead container ready
for collection. The product was then removed from the synthesizer and subjected to QC
analysis by analytical HPLC.
Animal studies: The biodistribution of the tracer was examined in mice. All studies were
approved by the Animal Ethics Committee of The University of Queensland in accordance
with the Australian Code for the Care and Use of Animals for Scientific Purposes.
PET/CT imaging protocol: Four healthy C57BL6J female mice aged 12 weeks were
anaesthetized with 2% isoflurane (IsoFlo, Abbott Laboratories) in a closed anesthetic induction
chamber. Animals were monitored using ocular and pedal reflexes to ensure deep anesthesia.
Anaesthetized mice, with a cannulated tail vein, were placed in an Inveon PET/CT scanner
(Siemens). Physiological monitoring was performed using an animal monitoring system (the
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BioVetTM system, m2m Imaging, Australia). Mice were injected with approximately 5 MBq of
18F-fluoro-diphenyl gallium dithiosemicarbazone solution in a volume of 150 µL via the tail
vein catheter in a slow bolus injection.
Dynamic PET data acquisition was performed for 60 min. The emission data were normalized
and corrected for decay and dead volume. The list-mode data were sorted into 54 dynamic
frames (12 x 10 s, 16 x 30 s, 20 x 60 s, 6 x 300 s time frames). The resulting sinograms were
reconstructed using a filtered back-projection and an ordered-subset expectation maximization
(OSEM2D) algorithm and analyzed using the Inveon Research Workplace software (IRW,
Siemens). Following the PET scans, the CT images of the mice were acquired through an X-
ray source with the voltage set to 80 kV and the current set to 500 µA. The scans were
performed using 360° rotation with 120 rotation steps with a low magnification and a binning
factor of four. The exposure time was 230 ms with an effective pixel size of 106 µm. The total
CT scanning process took approximately 15 min. The CT images were reconstructed using
Feldkamp reconstruction software (Siemens). CT and PET datasets of each individual animal
were aligned using IRW software (Siemens) to ensure good overlap of the organs of interest.
Using morphologic CT information, three dimensional regions of interest were placed over the
whole body, as well as individual organs of interest including the heart, kidney, lungs, liver,
and brain. Activity per voxel was converted to nCi/mL using a conversion factor obtained by
scanning a cylindrical phantom with a solution containing fluorine-18 of known activity to
account for PET scanner efficiency. Activity concentrations were then expressed as the
percentage of the decay-corrected, injected activity per cm3 of tissue, approximated as
percentage injected dose/g of tissue (%ID/g). At the end of the imaging sessions, the mice were
sacrificed, and the organs were surgically removed, weighed and placed in tubes. The tubes
were counted using a gamma counter (Perkin Elmer, Model 2480, Gamma Counter Wizard 2,
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Illinois, USA) and the results were expressed as % injected dose/g organ and % injected
dose/organ to confirm the data obtained with the PET/CT imaging study.
Results and Discussion:
Synthesis of gallium diphenyl dithiosemicarbazone chloride was achieved using a previously
published procedure 45,46. The diphenyl thiosemicarbazone was treated with sodium methoxide
in anhydrous methanol followed by the addition of a stoichiometric amount of gallium chloride
pre-dissolved in anhydrous methanol as shown in Scheme 1.
Subsequently, an exchange reaction was conducted to replace the chloride with nitrate, a better
leaving group. The heterogeneous phase transfer methodology using silver nitrate in methanol
is shown in Scheme 2. The nitrate complex obtained was further treated with [18F]F- in a
subsequent exchange to obtain the 18F-radiolabeled gallium complex of diphenyl
dithiosemicarbazone. HPLC purification and reformulation of the tracer was achieved by using
a Synthra automated module. Figure 1 shows the Graphical User Interface (GUI) of the Synthra
automated module used for the HPLC purification of the crude reaction mixture and subsequent
reformulation. As can be seen from figure 1 that certain alterations in the synthetic module for
the purification of the final radiolabeled compound was necessary. As explained before, radio
labelling was performed manually using heterogenous reaction of silver nitrate and gallium
ligand followed by reaction with 18F, this modification was essential. In other words, the whole
synthesis and purification was achieved using a semi-automated process.
The analytical HPLC chromatogram of the radiolabeled compound is shown in Figure 2. The
initial peak in the radio chromatogram is the [18F]F- followed by the second peak at 15.5 min,
corresponding to the radiolabeled product. The figures also depict the UV peak obtained for
the product. Caution should be exercised here not to mistake the large UV peak in the
chromatogram at 15-16 min for non-radioactive 19F-labelled gallium diphenyl
dithiosemicarbazone since under our experimental conditions, the retention time of the starting
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chloro- compound and the fluorine labelled complex was identical. Presumably, UV peak of
19
the F-labelled diphenyl gallium dithiosemicarbazone is very close to the unreacted chloro-
compound (see figure 2, lower panel). The semi preparative chromatogram is shown in Figure
3. The radioactive peak at 14 min was collected and was used for animal studies.
The QC analytical HPLC (Figure 4) shows that a clean 18F-radiolabeled diphenyl gallium
thiosemicarbazone complex was obtained at 15.3 min. The accompanying UV trace shows that
there are three peaks around that range. The second large peak has a shoulder peak at
19
approximately 15.25 min which we believe corresponds to the F-complex. Radiosynthesis
was repeated several times without difficulty and the radiochemical yield of each trial is
reported in Table 1. Ongoing work aims to enhance radiochemical yield.
In order to confirm the stability of the complex, the same batch radiolabeled complex used for
vivo studies was further evaluated for its stability. Figure 5 shows the HPLC chromatogram of
the complex after 2 hours of post injection. It is evident from the figure that the HPLC profile
showed no sign of decomposition of the complex demonstrating the stability of the radiotracer
under our experimental conditions. The absence of uptake in bone also confirms the stability
of this radiotracer.
Stability of the intermediate nitrate complex: The nitrate complex was stable when stored as
a solid under nitrogen atmosphere in a sealed vial. Aliquots from a single batch of the complex
stored under nitrogen atmosphere for weeks could be radiolabeled without difficulty.
Homogenous solution exchange with silver triflate: We also examined the use of silver triflate
as the homogenous exchange medium (Scheme 3). Silver triflate is soluble in organic solvents
whereas silver nitrate is insoluble in methanol. Although we were able to radiolabel the
compound, the yield of the product was considerably lower, perhaps due to the high reactivity
of the triflate for trace amounts of water in the solvents. It is possible that less-reactive silver
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salts like tosylate could improve this exchange methodology and it may be possible to use other
metal salts for future applications.
Biodistribution of 18F-diphenylgalliumdithiosemicarbazone: The biodistribution is similar to
that of blood flow tracers pyruvaldehyde bisthiosemicarbazone copper complexes 47. PET/CT
images of tracer uptake in a single mouse are shown in Figure 6.
Time activity curve obtained from healthy C57BL6J mice injected with 18F-thiosemicarbazone
is shown in Figure 7. At 60 min, the highest uptake was seen in the liver followed by the heart,
kidney, gut and brain. Rapid uptake (peak uptake approximately 2 min) with an injected
dose/gram of 2 to 30% was observed in individual organs including the brain, the latter
suggesting that the tracer crosses the blood-brain barrier. Other researchers have described that
pyruvaldehyde bisthiosemicarbazone copper complexes efficiently crosses the blood brain
barrier 15,27,28,45,46,48. Most notably, no significant radioactivity was observed in bone, which
suggests that the Ga-fluoride bond is stable in vivo. Biodistribution at 60 min is presented in
Table 2 and is consistent with the dynamic imaging data.
Conclusions:
This research builds on the use of complexes of group-13 metals to form high affinity
complexes with [18F]fluoride 49, driven with a goal for the development of lipophilic
radiolabeled complexes which may have applications of neuroimaging. Whilst Bhalla et al.40-
42 reported the preparation of the first Ga-18F complexes, they did not report the properties of
their complexes in vivo. This paper describes the first example of in vivo imaging of a Ga-18F
compound. Furthermore, our studies demonstrate that the Ga-18F bond is stable in vivo with
our thiosemicarbazone ligand. Whilst our current yields are low, the stability of the Ga-18F
bond in vivo suggests that Ga-18F complexes of other derivatives of thiosemicarbazones and of
other chelate frameworks should be explored.
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Acknowledgements: The research was funded in part by the Australian Research Council
Linkage Grant #LP130100703.
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Scheme 1. Synthetic scheme for the preparation of gallium diphenyl dithiosemicarbazone
complex.
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Scheme 2. Radiosynthesis of 18F radiolabeled diphenyl gallium dithiosemicarbazide using
exchange method.
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Scheme 3. Use of silver triflate as a homogenous solution exchange catalyst for introducing
fluorine-18 in diphenyl gallium dithiosemicarbazone complexes.
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Figure 1. GUI of Synthra RN Plus automated radio-synthesis module. The fitting circled in
red is manually disconnected to allow attachment of a disposable syringe containing the
crude compound.
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Figure 2. Analytical radio chromatogram (top) and UV chromatogram (below) of sample of
crude reaction mixture. Retention time of labelled compound is ~ 15.3 minutes.
Radiolabeling yield is approximately 6% based on peak areas of the radio chromatogram.
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Figure 3. Semi-preparative radio chromatogram of crude reaction mixture diluted 50:50 with
water. The peak at ~14 minutes was collected via valve V27 of the Synthra module for
further SPE purification/reformulation.
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Figure 4. Analytical radio chromatogram (top) and UV chromatogram (below) of purified
gallium 18F-compound. Retention time of labelled compound is ~ 15.3 minutes.
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Figure 5. Analytical radio and UV chromatograms of purified gallium 18F-compound at 2.5
hours post synthesis. The Radiochemical purity remains >99% demonstrating
stability. Retention time of labelled compound is ~15.3 minutes.
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50%ID/g
A
B
C
D
0%ID/g
Figure 6. PET/CT Imaging of a healthy C57BL6J mouse after intravenous injection of 18F-
thiosemicarbazone. A: 5min, B: 15min, C: 30min, D: 60min after injection of the radiotracer.
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35
30
25
20
15
10
5
Heart
Liver
Kidneys
Guts
Brain
0
0
5
10
15
20
25
30
Time (min)
35
40
45
50
55
60
Figure 7. Time activity curve obtained from healthy C57BL6J mice injected with 18F-
thiosemicarbazone (Mean ± SD, n=4)
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Table 1. Reagent quantities and radiochemical yield data. The non-purified product yield is
based on the % areas of the product peak compared to the [18F]F- peak of the radio-
chromatogram of a sample of the crude reaction mixture. The purified product yield is based
on the radioactivity trapped on the C18 Solid Phase Extraction (SPE) cartridge after the semi-
preparative HPLC fraction cut compared to the starting quantity of [18F]F-. For each
experiment, the chemical precursor was dissolved in 500 μL DMSO.
Starting
Non-corrected
Radiochemical Yield, Radiochemical
non-purified (%)
Non-corrected
K222
(mg)
Acetonitrile
(μL)
K2CO3
(mg)
Aqueous
Activity
[18F]F- (μL)
[18F]F- (MBq)
Yield, purified (%)
1.9
1.8
2.1
1.9
1.9
1.9
1.9
2.0
2.1
1.9
1.9
1.2
1.2
1.2
1.3
500
500
500
500
500
500
500
500
500
500
500
200
200
200
200
0.35
0.35
0.35
0.35
0.35
0.35
0.35
0.35
0.35
0.35
0.35
0.55
0.57
0.60
0.55
100
100
100
100
100
100
25
4400
4400
4000
4870
2740
1720
277
0.44
0.77
0.56
0.48
0.62
0.78
1.19
5.4
6.1
1.3
3.0
4.3
8.1
2.2
4.8
5.7
100
200
150
100
60
148
3100
806
466
568
100
10
740
81
15
82
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Table 2. Biodistribution 75 min after intravenous injection of 18F-thiosemicarbazone in 4
healthy C57BL6J Mice. Results have been decay corrected to the time of the injection of the
radiotracer and are expressed as Mean ± Standard Deviation (SD).
% Injected dose/organ
0.47 ± 0.09
% Injected dose/g of organ
4.37 ± 0.39
Heart
Lungs
Liver
0.82 ± 0.10
4.94 ± 0.43
16.9 ± 3.0
18.9 ± 3.1
0.20 ± 0.05
2.45 ± 0.48
Spleen
Kidneys
Gut
2.41 ± 0.31
5.39 ± 0.35
0.12 ± 0.03
9.50 ± 0.83
2.57 ± 0.13
0.27 ± 0.06
Brain
Blood
Muscle
13.9 ± 1.9
1.18 ± 0.18
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