Thermochromatographic separation of 45Ti and subsequent radiosynthesis of [45Ti]salan

Due to its favorable decay properties, the non-standard radionuclide 45Ti is a promising PET isotope for tumor imaging. Additionally, titanium complexes are widely used as anti-tumor agents and 45Ti could be used to study their in vivo distribution and metabolic fate. However, although 45Ti can be obtained using the 45Sc(p,n)45Ti nuclear reaction its facile production is offset by the high oxophilicity and hydrolytic instability of Ti4+ ions in aqueous solutions, which complicate recovery from the irradiated Sc matrix. Most available 45Ti recovery procedures rely on ion exchange chromatography or solvent extraction techniques which are time-consuming, produce large final elution volumes, or, in case of solvent extraction, cannot easily be automated. Thus a more widespread application of 45Ti for PET imaging has been hampered. Here, we describe a novel, solvent-free approach for recovery of 45Ti that involves formation of [45Ti]TiCl4 by heating of an irradiated Sc target in a gas stream of chlorine, followed by thermochromatographic separation of the volatile radiometal chloride from co-produced scandium chloride and trapping of [45Ti]TiCl4 in a glass vial at − 78 °C. The recovery of 45Ti amounted to 76 ± 5% (n = 5) and the radionuclidic purity was determined to be > 99%. After trapping, the [45Ti]TiCl4 could be directly used for 45Ti-radiolabeling, as demonstrated by the successful radiosynthesis of [45Ti][Ti(2,4-salan)].


Introduction
Discovery of the anticancer activity of cisplatin and its clinical introduction in the 1970s have spurred interest into metal based antitumor compounds with less side effects and increased effectiveness against a broad range of cancers [1,2]. Titanium(IV) complexes like budotitane, titanocene dichloride and their derivatives are effective against various cancer cell lines but failed in in vivo clinical trials [3][4][5][6], most likely due to their rapid (within seconds) hydrolysis under physiological conditions [7]. A more promising class of titanium-based drugs with hydrolytic half-lives in the range of hours is based on tetradentate diaminobis(phenolato) ligands (salans) [8]. Their titanium complexes selectively induce apoptotic cell death [9] and display strong antitumor properties in vitro and in tumorbearing mice [10,11]. Further studies on their distribution, uptake and mechanism of action rely on imaging techniques such as positron emission tomography (PET), which allows for non-invasive assessment of the biological fate of radiolabeled drugs while they distribute in vivo. The titanium isotope 45 Ti has a half-life of 3.1 h, a high positron branching ratio (β + = 84.8%) and low maximum positron energy (E β+max = 439 keV), negligible secondary gamma emission and a low β end point energy of 1.04 MeV, making it an ideal candidate for use in PET studies [12][13][14]. However, while the radiometal can be produced by transmutation of naturally monoisotopic scandium with low energy protons [15,16], 45 Ti radiochemistry is hampered by the high oxophilicity (θ = 1.0) and hydrolytic instability of Ti 4+ ions in aqueous environments, which necessitate the use of strongly acidic conditions [17,18]. Solid phase extraction of 45 Ti dissolved in acidic solutions by ion exchange chromatography is timeconsuming, cannot easily be automated and often results in non-reactive titanyl species that are unsuitable for production of titanium complexes [19,20]. A number of approaches have been proposed to circumvent these problems, which include the use of hydroxamate resins and oxalic acid elution [21], trapping of 45 Ti on a diol-functionalized resin followed by on-resin radiosynthesis [22] and continuous liquid-liquid extraction of the radioisotope with a guaiacol/anisole mixture to obtain a 45 Ti containing organic phase that can be used for radiolabeling [23,24]. Here, we describe an alternative, solvent-free "one-pot" method that is based on thermochromatographic separation of 45 Ti from an irradiated Sc target and consecutive radiotitanation of a salan ligand, thereby obviating the need for organic solvents, solid phase extraction or on-column chelation chemistry.

Analytical instrumentation
Gamma spectroscopy was performed with ORTEC gammaray spectrometers (AMETEK GmbH, Germany), which were energy and efficiency calibrated with certified radiation point sources (Co-60, Ba-133, Eu-152, Ra-226) from the Physikalisch-Technische Bundesanstalt (Germany). To monitor the separation process, a Geiger-Müller-Tube (Vacu-Tec GmbH, Germany) was used. . High-resolution mass spectrometry (HRMS) was performed using a hybrid linear ion trap FT-ICR mass spectrometer LTQ-FT Ultra (Thermo Fisher Scientific, Germany) equipped with a 7 T superconducting magnet. The electrospray ionisation (ESI) source was operated in the positive mode. Low-resolution mass spectrometry was measured using a Finnigan Automass Multi spectrometer (Thermoquest, Germany). 45 Sc concentrations in solution were determined by inductively coupled plasma mass spectrometry (ICP-MS) using an Agilent 7500 instrument with quadrupole mass analyzer and collision cell (Agilent Technologies, Germany). The instrument was operated with helium as collision gas to minimize spectral interferences by cluster ions. Quantification was performed by external calibration using Rh as the internal standard.

Radionuclide production
Titanium-45 was produced from discs of metallic scandium (0.35 ± 0.1 g) by the 45 Sc(p,n) 45 Ti nuclear reaction. As target material scandium of 99.99% purity was used (Smart-Elements GmbH, Austria). The material came in form of small ingots, which were rolled to plates of 0.6 mm. From those plates circular target of 13 mm were cut and put into a copper target holder to be irradiated at the solid target station of the BC1710 [30]. The target was irradiated with 8.2-16.9 MeV protons at 1.5 µA for 30 min using the Baby Cyclotron BC1710 at the INM-5 (Forschungszentrum Jülich). To minimize coproduction of 44 Sc (half-life: 3.9 h), 44m Sc (half-life: 58 h) and 44 Ti (half-life: 60 y), two Ni foils with a thickness of 125 µm each were used to degrade the proton energy to approximately 12 MeV. For initial optimization studies, irradiation was performed without degradation of the proton beam, so that 44 Sc and 44m Sc could be used to radiometrically monitor the separation process and determine the radiochemical purity of the product.

Radionuclide separation system
The separation set-up ( Fig. 1) consisted of a quartz glass reaction chamber that was connected to the main gas line via a Young valve and could be heated to temperatures of up to 1000 °C by a model MTF 12/39/250 heating oven (Carbolite Gero GmbH, Germany), a borosilicate glass tube condenser that could be heated to 400 °C, a receiving flask that could be cooled down to − 79 °C by an acetone/CO 2 cooling bath and a chlorine scrubbing system with 20% sodium hydroxide solution. Various gas mixtures were supplied to the setup by two mass flow controllers (Fig. 2). The ground glass joint connecting the reaction chamber and the condenser was sealed with a polytetrafluoroethylene sealing ring to obviate the need for high vacuum grease which could have absorbed or contaminated the product. In addition, the upper part of the receiving flask was equipped with a septum, which allowed for addition of reagents during the radiosynthesis that was performed immediately after the separation process.

Thermochromatographic separation of 45 Ti from scandium
For isolation of titanium-45, the irradiated Sc matrix was placed into the reaction chamber, which was flushed with helium (125 mL/min) and heated to 900 °C. When the reaction temperature was reached, the chamber was flushed with a mixture of chlorine (15 mL/min) and helium (110 mL/ min) gas to form the halides [ 45 Ti]TiCl 4 and ScCl 3 respectively. As the gaseous mixture of metal chlorides reached the condenser, ScCl 3 (boiling point ~ 975 °C) resublimed while [ 45 Ti]TiCl 4 (boiling point ~ 136 °C) passed further and condensed in the receiving flask. A Geiger-Müller counter was used to monitor the separation process. When the collection of [ 45 Ti]TiCl 4 was complete, the addition of chlorine and heating were discontinued, the temperature of the receiving  flask was raised to − 30 °C and the system was flushed with helium for 10 min to remove excess chlorine. Optimal reaction conditions (temperature, chlorine concentration and gas flow) were determined experimentally as described in more detail in the results section and supporting information. 6′-((ethane-1,2-diylbis((2-hydroxyethyl) azanediyl))bis(methylene))bis(2,4-dimethyl-phenol) (2,4-salanH 4 ) 740 mg (1 eq, 5.0 mmol) 1,2-bis(2-aminoethoxy)ethane, 122 mg 2,4-dimethylphenol (2 eq, 10.0 mmol) and 500 mg paraformaldehyde (3.3 eq, 16.7 mmol) were dissolved in methanol and refluxed for 12 h. The organic solvent was removed under reduced pressure and the crude product recrystallized from diethylether/petrolether to obtain the target compound as white crystals. (251 mg, yield: 12%

Radiosynthesis of [ 45 Ti][Ti(2,4-salan)]
2 mg 2,4-salanH 4 and 20 µl of DIPEA in 2 mL anhydrous THF were added to the receiving flask containing the isolated [ 45 Ti]TiCl 4 and allowed to react for 5 min at − 30 °C, after which the mixture was analyzed by HPLC. For purification of the radiolabeled complex, the reaction mixture was diluted with H 2 O (30 mL) and loaded onto a SepPak C18 RP cartridge (Waters GmbH, Germany). The cartridge was washed with a mixture of H 2 O/MeOH/CH 3 COOH (4/1/0.005 mL) and the product eluted with MeOH (3 mL).

Results and discussion
Routine production of 45 Ti by proton bombardment of natural scandium resulted in high radionuclidic purity and good yields. The latter amounted to about 290 MBq for a typical bombardment with 12 MeV protons and 1.5 µA current for 30 min of beam time. When choosing 16.9 MeV incident proton energy, a typical bombardment of 1.5 µA for 30 min produced a batch yield of 360 MBq and the product was composed of 87% 45 Ti, 12% 44 Sc and 1% 44m Sc. These production yields are comparable to those reported by Vavere et al. [19]. For isolation of the radiometal and formation of radiolabeled complexes, we developed a solvent-free "onepot" procedure that involves (1)

Preparation of [ 45 Ti]TiCl 4 by high-temperature chlorination of irradiated Sc
To avoid the formation of titanyl species, [ 45 Ti]TiCl 4 for PET radiochemistry has almost exclusively been produced by dissolving irradiated scandium targets with concentrated hydrochloric acid. Recovery of the radiometal chloride from acidic solutions involves arduous separation chemistry, which has limited a more widespread application of 45 Ti for PET imaging. To circumvent these problems, we investigated high-temperature chlorination, which has long been used for commercial TiCl 4 production from rutile (TiO 2 ) or ilmenite (FeO-TiO 2 ) feedstocks [25]. To this end, we first examined whether [ 45 Ti]TiCl 4 could be produced and separated from the irradiated target under conditions that minimize formation of scandium chlorides. However, preliminary experiments with non-irradiated targets treated with 100% Cl 2 demonstrated that formation of ScCl 3 starts to occur at temperatures above 600 °C which is still below the 850-1050 °C typically used for chlorination of titanium ores [25]. Also, no formation of [ 45 Ti]TiCl 4 was observed when an irradiated target was chlorinated at 550 °C. When the temperature was increased to 750 °C, some formation of [ 45 Ti]TiCl 4 took place but isolation was hampered by an exothermic reaction of the scandium with Cl 2 , which contaminated the whole separation apparatus. To avoid this exothermic reaction, a series of optimization experiments was performed with a fixed reaction temperature of 900 °C and a total gas flow rate of 100 mL/min while varying the concentration of chlorine. For these experiments, irradiation of the Sc target was deliberately performed without degradation of the proton beam, so that 44 Sc and 44m Sc could be used to radiometrically monitor the separation. As illustrated in Table 1, 45 Ti recovery under these conditions was maximal (53%) when the chlorine concentration was 12%. In a second set of optimization experiments, temperature and chlorine concentration were fixed at 900 °C and 12% while the total gas flow rate was varied ( Table 2). The results showed that highest recovery rates could be obtained with total gas flow rates between 100 and 150 mL/min, whereas recovery decreased at lower or higher rates (Table 2). Finally, we examined the influence of reaction temperature on recovery of 45 Ti with the total flow rate fixed to 125 mL/min and a Cl 2 concentration of 12%. The recovery of [ 45 Ti]TiCl 4 decreased as the temperature was lowered to 850 °C (Table 3), presumably because ScCl 3 formation on the surface of the target prevented reaction of the radiometal with Cl 2 . Better results (53% recovery) were obtained at 900 °C, a temperature at which sublimation of ScCl 3 was sufficient to prevent its accumulation on the surface of the target. If the temperature was further increased to 1000 °C, the recovery yield decreased again by 13%, most likely reflecting the thermal decomposition of [ 45 Ti]TiCl 4 at that temperature [26,27].

Thermochromatographic separation and purification of [ 45 Ti]TiCl 4
As noted above and consistent with previous reports [28,29], appreciable transition of ScCl 3 to the gas phase was observed at temperatures above 850 °C, requiring its removal from the gaseous mixture of metal chlorides. This was initially attempted with fritted glass, which proved to have several disadvantages. Apart from loss of product on the large surface area of the frit, re-sublimation of ScCl 3 tended to clog the system, which could potentially result in dangerous pressure build-up. Therefore, a simple condenser made of a 100 cm borosilicate spiral coil that was heated to 400 °C and served as the vapor-condensate path was used instead. In initial experiments with argon as the inert gas, no separation could be achieved since ScCl 3 was transported to the receiving flask. In contrast using helium as the inert gas, ScCl 3     Finally, the optimized reaction conditions were used for the separation of pure 45 Ti, produced by degrading the proton energy for irradiation of the Sc target to approximately 12 MeV (see Experimental section). This allowed precise quantification of 45 Ti recovery by simply dividing the recovered activity of 45 Ti by the total starting activity. On average, recovery of 45 Ti amounted to 76 ± 5% (n = 5). The separation process was conducted within 2 h, corresponding to a non-decay-corrected (n.d.c.) recovery of 48 ± 3% (n = 5) ( Table 4). The amount of non-radioactive Ti was analyzed by ICP-MS to be less than 1 µg/L in the final reaction volume.

Radiosynthesis of [ 45 Ti][Ti (2,4-salan)]
To confirm that the isolated [ 45 Ti]TiCl 4 can be directly employed for radiolabeling, it was reacted with the hexadentate ligand 2,4-salan to produce a [ 45 Ti][Ti(2,4-salan)] complex (Fig. 3). To this end, 2,4-salan and DIPEA were dissolved in dry THF, added to the receiving flask containing the purified [ 45 Ti]TiCl 4 and allowed to react for 5 min at − 30 °C. HPLC of the reaction mixture with co-injection of the non-radioactive reference compound confirmed the identity of the product and the absence of radioactive byproducts (Fig. 4). However, radio-TLC revealed up to 50% of an unidentified byproduct, presumably [ 45 Ti]TiO 2 , which may have formed due to traces of H 2 O present in the reaction mixture. From the starting activity used for this reaction, 50% could be recovered from the receiving vessel for further purification. The radiochemical yield (RCY) obtained after isolation of the radiocomplex via solid phase extraction amounted to 15 ± 7% (n = 7).

Conclusions
In this work a radioseparation method of no-carrier-added [ 45 Ti]TiCl 4 from Sc is described. 45 Ti was produced by proton irradiation of a metallic Sc target followed by thermochromatographic separation in a chlorine gas stream. The separated [ 45 Ti]TiCl 4 could be used directly for the preparation of the radiometal complex [ 45 Ti][Ti(2,4salan)] in radiochemical yields of 10-20%. This simple separation technique obviates the need for complex separation chemistry and may contribute to a more widespread application of 45 Ti-labeled compounds in imaging applications. technical assistance, and to Mr. M. Holzgreve and Mr. I. Montag for all cyclotron-irradiations. Further thanks go to F. Neumaier for his assistance in the preparation of this manuscript.
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