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

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

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 1950^{s 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. ^34-39and Bhalla et al. ^40-43using 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

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

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.

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/K₂CO₃was 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/K₂CO₃and 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

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

BioVet^TMsystem, 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 cm³of 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,

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

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

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-

⁴²reported 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.

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.

Scheme 2. Radiosynthesis of ¹⁸F radiolabeled diphenyl gallium dithiosemicarbazide using

exchange method.

Scheme 3. Use of silver triflate as a homogenous solution exchange catalyst for introducing

fluorine-18 in diphenyl gallium dithiosemicarbazone complexes.

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.

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.

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.

Figure 4. Analytical radio chromatogram (top) and UV chromatogram (below) of purified

gallium ¹⁸F-compound. Retention time of labelled compound is ~ 15.3 minutes.

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.

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.

35

30

25

20

15

10

5

Heart

Liver

Kidneys

Guts

Brain

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)

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

2.0

2.1

1.9

1.2

1.3

500

200

0.35

0.55

0.57

0.60

0.55

100

25

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

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

Article Doi

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

DOI: 10.1002/jlcr.3746

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