Fully Automated Synthesis of Novel TSPO PET Imaging Ligand [18F]Fluoroethyltemazepam

Introduction: Benzodiazepines, including temazepam are described as TSPO antagonists. In fact, TSPO was initially described as a peripheral benzodiazepine receptor (PBR) with a secondary binding site for diazepam. TSPO is a potential imaging target of neuroinflammation because there is an amplification of the expression of this receptor. Objectives: Herein, we developed a novel fluorinated benzodiazepine ligand, [18F]Fluoroethyltemazepam ([18F]F-FETEM), for positron emission tomography (PET) imaging of translocator protein (18 kDa). Methods: [18F]F-FETEM was radiolabelled with an automated synthesizer via a one-pot procedure. We conducted a [18F]F-aliphatic nucleophilic substitution of a tosylated precursor followed by purification on C18 and Alumina N SPE cartridges. Quality control tests was also carried out. Results: We obtained 2.0–3.0% decay-uncorrected radiochemical activity yield (3.7% decay-corrected) within the whole synthesis time about 33 min. The radiochemical purity of [18F]F-FETEM was over 90% by TLC analysis. Conclusions: This automated procedure may be used as basis for future production of [18F]F-FETEM for preclinical PET imaging studies.


Introduction
Context and background: Translocator protein TSPO is a protein located on the outer mitochondria membrane mainly expressed in the brain in glial cells. Mitochondria are involved in several vital and detrimental processes and mitochondrial dysfunctions are related in a variety of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and psychiatric condition such as anxiety and bipolar disorder [1].
TSPO was initially described as a peripheral benzodiazepine receptor (PBR) with a secondary binding site for diazepam; however, subsequent research has found that this receptor is expressed throughout whole body. In humans, translocator protein is encoded by the TSPO gene and belongs to the tryptophan-rich family of sensory proteins [2].
TSPO-binding ligands showed influence in a variety of cellular functions (i.e., cholesterol transport and steroid hormone synthesis, mitochondrial permeability, apoptosis, proliferation, tumor genesis and inflammation [3][4][5]. In the brain, TSPO increase expression was found in activated microglia during neuroinflammation and immunosuppressive action of the TSPO ligands make TSPO an attractive target for diagnostic imaging [6]. TSPO translocator protein is an 18 kDa protein with five strongly hydrophobic transmembrane domains, found mainly on the outer mitochondrial membrane of microglia [7]. This receptor interacts with StAR (steroidogenic acute regulatory protein) for the transport of cholesterol into mitochondria [3]. In the last few years, numerous PET imaging radiotracers specific for TSPO have been synthesized and reported in the literature labeled with carbon-11 and fluorine-18 [8].
Over the last few years, numerous ligands (and radioligands) for TSPO have been synthesized and reported in the literature [9]: i.
TSPO antagonists, including benzodiazepines temazepam, showed a significant antagonistic properties based on cell proliferation assays. Although benzodiazepines, such as temazepam, are molecules well known in literature and have been used for decades in clinical practice, clinical application as antagonists of TSPO is still in an initial phase (in vitro) [10].
PK11195 isoquinoline was originally described as a compound that partially displaced some benzodiazepines such as diazepam. In fact, this compound has receptor affinity for peripheral benzodiazepine receptors. Furthermore, PK11195 can be found in mitochondrial fractions of brain extracts and in mitochondria-free erythrocytes.
Clinical use of this tracer for PET imaging is based on three observations: only minimal PK11195 binding is shown in healthy patients; in central nervous system diseases, in neuroinflammatory conditions, binding is found particularly in activated microglia; radiolabelling with carbon-11 allows PET imaging.
Therefore, other radiotracers have also been developed with preclinical and clinical studies, some of which use fluorine-18 as radioisotope which has a longer half-life, compared to carbon-11 (109 min compared to 20 min for carbon-11, according to database of the National Nuclear Data Center (NNDC) at Brookhaven National Laboratory, Upton, NY, USA).
Unknown factors/problems: However, several practical problems arise with TSPO radiotracers: not distinguishing pro-and anti-inflammatory responses, low signal-tonoise ratio, low variation of dynamic response during neurodegenerative diseases, TSPO polymorphisms.
These problems appear to have been reduced and/or eliminated with second and third generation radiotracers; however, there is still no possible differentiation between the M1 (neurotoxic) and M2 (neuroprotective) genotypes of microglia. Furthermore, even if 3D pentameric structure of the TSPO has been revealed, the role of this receptor is not yet clear, as well as the influence of receptor upregulation in immune reaction is not clear. New studies have shown that TSPO radiotracers (especially first and second-generation ones) show three binding affinity patterns derived from the rs6971 polymorphism: high affinity ligands (HABs), mixed affinity ligands (MABs) and low affinity ligands. (LABs) [12]. PET signal provided in MAB and LAB patients leads to a significant underestimation TSPO expression. However, it seems that the third generation TSPO radioligands are not sensitive to the rs6971 polymorphism, such as [ 18 F]flutriciclamide ([ 18 F]F-GE180); moreover, it would seem that the lesion/background ratio is higher than the other TSPO tracers [13,14].
Experimental approach/major findings: In this paper, we introduce a new type of TSPO radiotracer (3S)-7-chloro-3-[ 18 F]fluoroethyl-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2-one, [ 18 F]Fluoroethyltemazepam ([ 18 F]F-FETEM) radiolabelled with an automated synthesizer via a one-pot procedure. Validated methods reported in literature for benzodiazepines radiolabeling (such as [ 18 F]Flumazenil) make use of purification methods by semipreparative HPLC. These synthesis methods involve an increase in production times, which can even reach 80 min, with a yield corrected by decay of 15%. Herein, proposed synthesis and purification method achieved a balance between reduction of synthesis times, removal of impurities using SPE cartridges, use of a versatile synthesis module with a noninvasive cleaning procedure and without carryout of products from previous processes.
Implications: [ 18 F]F-FETEM can extend our understanding on TSPO implication in neurodegenerative diseases.

Results
In contrast, to procedure described in literature, we were able to apply a standardfluorination technique that involved preparation of dry K[ 18 F]/kryptofix complex following nucleophilic displacement reaction at 120 • C for 5 min in anhydrous DMSO. In order to optimize reaction conditions, temperature, time and solvent were investigated. An

Discussion
The objective of our research was synthesis of the target compound [ 18 F]F-FETEM and his corresponding p-toluenesulfonyl precursors (Table 1), required for fluorine-18 labelling. The increased clinical need for TSPO radiotracers prompted us to design an automated device for [ 18 F]F-FETEM synthesis. Table 1. Summary of TSPO PET tracers most commonly described in literature.

PET Tracer
Chemical Structure TSPO Generation Reference First generation [15] [ 18 Second genera- [17] Third generation [16] Second generation [20] For the automated synthesis of [ 18 F]F-FETEM we used a custom-modified commercially available automated synthesis module for nucleophilic 18 F-fluorinations TRAC-ERlab™ FXFDG, which is similar to the TRACERlab™ FXN synthesis module, which has been shown to be a versatile synthesis platform for the production of a range of different 18 F-labelled radiopharmaceuticals [21].
The radiolabelling of [ 18 F]F-FETEM was achieved in one step synthesis by classical [ 18 F]fluoride nucleophilic substitution of the p-toluenesulfonyl precursor using potassium carbonate and Kryptofix ® 222 at 100 °C for 5 min (Figures 1 and 2)  Second generation [20] For the automated synthesis of [ 18 F]F-FETEM we used a custom-modified commercially available automated synthesis module for nucleophilic 18 F-fluorinations TRACER-lab™ FXFDG, which is similar to the TRACERlab™ FXN synthesis module, which has been shown to be a versatile synthesis platform for the production of a range of different 18 F-labelled radiopharmaceuticals [21].
The radiolabelling of [ 18 F]F-FETEM was achieved in one step synthesis by classical [ 18 F]fluoride nucleophilic substitution of the p-toluenesulfonyl precursor using potassium carbonate and Kryptofix ® 222 at 100 • C for 5 min (Figures 1 and 2) in a closed reactor under helium flow.  Owing to a high reaction temperature (100 • C) solvent (DMSO) was used with no evaporation during the reaction because of short reaction times (5 min). The Sep-Pak purification provided a simple separation of [ 18 F]F-FETEM from radiochemical impurities. After purification and formulation of the radiotracer, results from this study provide evidence an equivalent radiochemical yield (non-decay-corrected) of 2.0-3.0% (3.7% decaycorrected) of [ 18 (Figures 3-5). Since this TLC chromatographic system has already been verified and reported in literature [22], we can hypothesize that other radiochemical impurities obtained from TLC chromatograms are benzodiazepine molecules shown in Figure 6.
These impurities derive from radiofluorination directly on benzodiazepine ring, in one case in position 3 (where the leaving group would be OH-EtTos) in another in position 6 (where the leaving group would be a chlorine atom). Radioimpurities do not increase during the stability period verified (3 h). According to preclinical studies data reported for TSPO radiotracers in the literature, this stability of 3 h allows us to perform in vivo biodistribution studies. Furthermore, radioimpurities do not increase in three hours and no decomposition products were found in the three hours analyzed so it allow to perform a longer stability study (6 h). Plus, in vivo preclinical studies are more feasible by using the fluorine-18 radioisotope, compared to carbon-11 (109 min compared to 20 min for carbon-11).    Our study expands on work of Barresi et al. [9] of TSPO ligands as potential therapeutic or diagnostic tools. In fact, in their work, benzodiazepines such as temazepam are not reported as ligands. In the literature, TSPO antagonists, including benzodiazepines temazepam, are reported in vitro but no benzodiazepine ligands have ever been used for PET imaging [10,23]. One potential limitation of future studies is that [ 18 F]F-FETEM binds TSPO and GABA receptors at the same time. In addition, we cannot define a yield optimization by varying the amount of precursor, including adjustments of the reaction temperatures and times. In the preclinical production of [ 18 F]F-FETEM, we encountered problems with lipophilicity of the [ 18 F]F-FETEM. The relatively low radiochemical yield (RCY) is due to radioactivity losses in incomplete radiochemical reactions, as well as to a lesser extent to side reactions. There is room for further optimization. However, we are able to produce sufficient amounts of [ 18  In conclusion, our results suggested a new type of benzodiazepine radioligands for TSPO never previously described. However, further studies are necessary to validate [ 18 F]F-FETEM in vivo with neurodegenerative diseases animal models.
Temazepam (2 g, 0.00665 mol), solubilized in DMF (10 mL), was added to thionyl chloride (3.95 g, 0.0332 mol) in a stoichiometric excess of 5 equiv. Reaction mixture was then kept under electromagnetic stirring at 0 • C whole night. Subsequently, reaction mixture was evaporated under reduced pressure to remove solvent and excess reactive and the product thus obtained (2.393 g, 0.0074 mol) was used without further purification in the subsequent reaction step.
Ethylene glycol (20 mL) was previously heated to a temperature of 50 • C to which the previously obtained chlorinated intermediate was added. The reaction mixture thus obtained was kept at 50 • C for 15 min. and was cooled to room temperature and it was left for further 4 h under electromagnetic stirring. Subsequently, ice was added to the reaction mixture and an extraction was carried out using chloroform. The organic phase thus obtained was back-extracted with water, anhydrified with anhydrous Na 2 SO 4 , filtered and evaporated. The intermediate thus obtained (2.775 g, 0.00805 mol) was used without further purification in the subsequent reaction step in which it was solubilized in anhydrous DCM (20 mL). To this solution were added tosyl chloride (1.726 g, 0.00905 mol, 1.12 equiv.), DMAP (0.393 g, 0.00321 mol, 0.4 equiv.), TEA (0.814 g, 1.12 mL, 0.00804 mol, 1 equiv.). Resulting mixture was kept under electromagnetic stirring at room temperature overnight. Reaction mixture was evaporated and residue was purified on a silica gel chromatographic column using ethyl acetate/hexane 7:3 v/v as eluent mixture. Finally, the final product thus obtained was recrystallized from hexane and identified by 1 H-NMR and 13 C-NMR nuclear magnetic resonance spectroscopy and ESI-MS mass spectrometry.
Addition of ethylene glycol (20 mL at 50 • C) to the reactor vial 4.
Reaction at 50 • C for 15 min and 4 h at room temperature under magnetic stirring 5.
Evaporation of organic phase

[18.F]Fluoride Production
Oxygen-18 enriched water in a silver target was irradiated with 9.6 MeV protons produced with a MINItrace cyclotron. The beam current was 40 µA for 30 min. After the irradiation, the target water with [ 18 F]Fluoride was transferred in a target vial of synthesizer via a helium flow at 60 psi.

[18.F]F-FETEM Production
The automated synthesis was controlled and monitored from PC and comprised the following automated steps (Table 3) In isolator a QC sample (1 mL) was collected into another sterile pyrogen-free glass vial previously labelled.

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After obtaining the QC sample, all filters and needles were removed from the endproduct vial and the radioactivity was measured.

Conclusions
Here, we described the development of an automated synthesis one-step one-pot reaction for preclinical production of [ 18 F]F-FETEM in a single-reactor TRACERlab™ FXFDG synthesis module. Moreover, the entire process is straightforward and this user-friendly device and process provides adequate quantities of [ 18 F]F-FETEM with low RCY for preclinical studies.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.