Rapid Aqueous Late‐Stage Radiolabelling of [GaF3(BnMe2‐tacn)] by 18F/19F Isotopic Exchange: Towards New PET Imaging Probes

Abstract A simple and rapid method for 18F radiolabelling of [GaF3(BnMe2‐tacn)] by 18F/19F isotopic exchange is described. The use of MeCN/H2O or EtOH/H2O (75:25) and aqueous [18F]F− (up to 200 MBq) with heating (80 °C, 10 min) gave 66±4 % 18F incorporation at a concentration of 268 nm, and 37±5 % 18F incorporation at even lower concentration (27 nm), without the need for a Lewis acid promoter. A solid‐phase extraction method was established to give [Ga18F19F2(BnMe2‐tacn)] in 99 % radiochemical purity in an EtOH/H2O mixture.

Fluorine-18 is the most widely utilised radioisotope in positron emission tomography (PET) imaging owing to its physical and nuclear characteristics:the short but manageable half-life (ca. 110 min), the short positron linear range in tissue (2.3 mm), the lack of side emission (97 %d ecay by positron emission), the low energy of the positron (E b max = 635 keV), and the wide availability of cyclotrons for its production. The 18 Fh alf-life is sufficient to allow for ac ertain degree of manipulation of the synthesis,p rovided that the radiolabelling occurs in the later stages of the synthesis (ideally in the final step). Them ost commonly used PET radiotracers are organic molecules where the radioactive fluorine atom is attached to ac arbon atom. Their generation often requires multistep syntheses and/or purification after the labelling step,w hich can be time-consuming and inefficient. This has driven recent work by several groups to investigate the 18 F radiolabelling properties of inorganic molecules,w here the strong bond between (typically) am ain-group element and fluorine can be exploited to enable fast late-stage radiolabelling.O ther important aspects to be considered are pH tolerance and the temperature required for radiolabelling as these will significantly influence the compatibility with biomolecules.A ni deal target for 18 Fr adiolabelling would be am ethod consisting of as ingle step where the [ 18 F]F À target water is introduced directly without further purification, at very low precursor concentration (e.g., 10 nm), without the need for post-labelling purification, giving ap roduct with high molar activity that is stable in the formulation matrix.
Ar ecent review from Gabbaï and co-workers describes some of the key advances in the development of Group 13 element based tracers towards PET applications. [1] Within the Group 13 elements,b oron has the highest bond dissociation energy with fluorine (> 730 kJ mol À1 ), [2] and after carbon, it has been the most studied element for PET applications. Several different types of molecules have been successfully radiolabelled with 18 F, including aryl trifluoroborates, [3][4][5] zwitterionic onium trifluoroborates, [6][7][8] and BODIPY-based dyes. [9,10] Typically,r adiofluorination is achieved by either converting aboronic ester moiety into afluoroborate species or by an isotopic exchange reaction. Very recently,t he development of [ 18 F]BF 4 À and [ 18 F]CF 3 SO 3 À as PET probes for imaging the sodium iodide symporter has been reported. [11][12][13] Work on the "A l-F" system developed by McBride and co-workers demonstrated that radiofluorination can be achieved by heating AlCl 3 ,[ 18 F]F À ,a nd ap entadentate NOTAderived ligand together in aqueous solution at pH 3.9-4.2 and 100 8 8C( NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid). [14] This was an important breakthrough, providing the first example of ametal chelate system for [ 18 F]F À capture in water, and exploiting coordination chemistry to determine fluoride incorporation. Ar elated gallium species was reported subsequently;i nt his case,t he [Ga 18 F(L)] moiety (L = 1-benzyl-1,4,7-triazacyclononane-4,7-dicarboxylate) was formed readily by radiofluorination of the preformed chloride complex [GaCl(L)] in aqueous MeCN under mild conditions. This 18 F-radiolabelled complex is stable to at least pH 6, but unlike the Al-F system, exhibits reduced stability in phosphate buffered saline (PBS) and human serum albumin (HSA) at pH 7.4. [15] This instability at higher pH was attributed, at least in part, to the lower stability of the carboxylate bonds to Ga III versus Al III .
In 2014, we reported as eries of trifluoride complexes of aluminium, gallium, and indium with neutral tridentate tacnbased macrocycles,i ncluding [MF 3 (BnMe 2 -tacn)] (M = Al, Ga, In;B nMe 2 -tacn = 1-benzyl-4,7-dimethyl-1,4,7-triazacyclononane), as potential [ 18 F]F À carrier molecules.N otably, these trifluoro complexes are extremely stable in water. [16] Furthermore,w ed emonstrated that the trichloro analogue, [GaCl 3 (BnMe 2 -tacn)],can be radiofluorinated easily at room temperature and close to neutral pH, by treating an MeCN/ In the fluorination of the metal trichloro species,t he strength of the MÀFb onds being formed undoubtedly provides as ignificant thermodynamic driving force for the rapid introduction of F À .Itisalso clear that the stability of the resultant radiofluorinated metal complexes in competitive media (PBS or serum) is also subtly dependent upon the choice of metal ion acceptor and any co-ligands present in the metal coordination sphere. [15] Whilst radiofluorination was readily achieved for [MCl 3 (BnMe 2 -tacn)] (M = Ga, Al) at 2.6 mm concentration, [16,17] to offer real prospects for the development of av iable imaging probe for PET based upon this system, it is desirable for the 18 Fincorporation to be efficient at lower, that is,n anomolar concentration. However,w ef ound that [MCl 3 (BnMe 2 -tacn)] did not undergo radiofluorination at 260 nm (0.1 mg mL À1 )c oncentration. We surmised that this could be due to the hydrolytic sensitivity of the M À Cl groups in [MCl 3 (BnMe 2 -tacn)],r esulting in competition between slow hydrolysis and Cl/F exchange under the labelling conditions. [16] In view of the apparent resistance of [GaF 3 (BnMe 2 -tacn)] to hydrolysis,w ef irst sought to probe the kinetic stability of this (inactive) trifluoro complex in aqueous solution under av ariety of conditions,before exploring the possibility of using 18 F/ 19 Fisotopic exchange reactions to produce the radiofluorinated product more conveniently and, preferably,using less material.
Am ore convenient synthesis of (inactive) [Ga 19 F 3 (BnMe 2 -tacn)] was established as an alternative to the hydrothermal method that we described previously. [16] Then ew method is based on the direct reaction of the molecular [GaF 3 (dmso)-(OH 2 ) 2 ]c omplex with BnMe 2 -tacn in CH 2 Cl 2 at room temperature,g iving the characteristic IR and 1 H, 19 Figure S4);t he strong affinity of [MF 3 (R 3 -tacn)] to alkalimetal cations has been demonstrated previously. [18] Solution 19 FNMR studies confirmed that [GaF 3 (Me 3tacn)] is very stable in water (spectrum unchanged) at elevated temperature (80 8 8C), even after several hours.T his is in contrast to the stability of [GaCl 3 (RMe 2 -tacn)] (R = Me or Bn), which hydrolyses within minutes when small amounts of water are added to as olution of the complex in MeCN at room temperature. [16] Thet rifluoro complex also shows very good pH tolerance,w ith no detectable degradation between pH 4and 11, and similarly,the spectra were unchanged upon addition of at enfold excess of (potentially) competitive Previous work by the group of Schirrmacher [19] on silicon fluoride based systems as well as by the groups of Blower, [20] Gabbaï, [6] and Perrin [21] on boron fluoride systems had demonstrated the value of 18 Figure 1a nd in the Supporting Information, Figures S5 and S6. Thei dentity of the radiolabelled product was confirmed with the corresponding UV traces (Figures S11-S13). Using the relatively lowactivity [ 18 F]F À employed in this work (ca. 200 MBq), the molar activity [22] determined for the 27 nm precursor concentration was ca. 675 MBq mmol À1 . As imple purification procedure using ah ydrophilic lipophilic balanced (HLB) solid-phase extraction (SPE) cartridge has also been established (see the Experimental Section), which gave the desired complex with ar adiochemical purity of about 99 %.
This method was also adopted successfully at an even lower (27 nm)p recursor complex concentration, resulting in 37 AE 5% 18 Fincorporation under similar labelling conditions (Table 1). This value corresponds to ad ecrease in the concentration of two orders of magnitude compared to the radiofluorination of [MCl 3 (BnMe 2 -tacn)] by Cl/F exchange reported previously.
Theradiochemical stability of purified [Ga 18 F 19 F 2 (BnMe 2tacn)] was also monitored over time (typically 2-3 h) for as eries of samples.A fter SPE purification with an HLB cartridge,w ef ound that the RCP had decreased to between 88 %a nd 77 %a fter 120 min at room temperature through loss of [ 18 F]F À from the radiolabelled product. An alternative purification procedure based on the use of an HLB cartridge followed by an alumina cartridge (at t = 0min) gave similar results,whereas cooling the purified [Ga 18 F 19 F 2 (BnMe 2 -tacn)] solution to À20 8 8Cled to an RCP of approximately 93 %after 4h( Figure S7).
As the DG value for the isotopic exchange is approximately zero,and some (ca. 4%)[ 18 F]F À incorporation is seen at room temperature (with much higher incorporation at 80 8 8C; Table 1), the following equilibrium reaction must apply: Theeffect of added fluoride or chloride was tested by the addition of either a1 0% K 19 Fs olution or a0 .9 %s aline solution to purified [Ga 18 F 19 F 2 (BnMe 2 -tacn)] at 25 8 8C. These had no significant effect on the RCP over about 2h.T hese results are consistent with the isotopic exchange proceeding through ad issociative mechanism;t hat is,i ti sf irst order in [GaF 3 (BnMe 2 -tacn)] and independent of the concentration of the entering ligand. Performing the radiofluorination in DMSO,amuch more competitive (strongly coordinating) solvent, leads to as ignificant drop in the RCY,w hich is also consistent with ap redominantly dissociative mechanism at the distorted octahedral Ga III complex that proceeds via afive-coordinate intermediate. [23] Te sting the stability of the radiolabelled product in 90 % human serum albumin (HSA)/10 %EtOH gave RCPs of 97 % at t = 0min and 83 %a tt = 120 min ( Figure S8). Thep ossibility of radiolysis leading to adecrease in RCP over time was also investigated, both by formulating the purified radiolabelled product in 10 %E tOH/PBS and by the addition of ascorbic acid to the purified radiolabelled product;n either had any appreciable effect (Figures S9 and S10).
In summary,w eh ave described the first 18 F/ 19 Fi sotopic exchange on ametal chelate system. Thenew method leads to high 18 Fi ncorporation by using [ 18 F]F À target water directly from the cyclotron and without the need for aL ewis acid promoter. Furthermore,wehave shown that the method also allows for the concentration of the [GaF 3 (BnMe 2 -tacn)] used for the radiofluorination to be scaled down by at least two orders of magnitude (27 nm,0.01 mg), which represents avery significant decrease in the quantity of material needed compared to the Cl À / 18 F À exchange reaction that we reported previously. [16] Ther esults reported here suggest that [Ga 19 F 3 (BnMe 2tacn)] offers ap romising basis for the development of new PET probes.F uture work will explore this system further 1) by using computational and experimental work to determine the effects that parameters such as the choice of the Group 13 metal and altering the steric protection around the M À Fgroups have on the 18 Fincorporation, and 2) through the conjugation of peptides (via the benzyl pendant group) to the most promising candidates to evaluate them as radiotracers in biodistribution studies.