Revisiting the Radiosynthesis of [18F]FPEB and Preliminary PET Imaging in a Mouse Model of Alzheimer’s Disease

[18F]FPEB is a positron emission tomography (PET) radiopharmaceutical used for imaging the abundance and distribution of mGluR5 in the central nervous system (CNS). Efficient radiolabeling of the aromatic ring of [18F]FPEB has been an ongoing challenge. Herein, five metal-free precursors for the radiofluorination of [18F]FPEB were compared, namely, a chloro-, nitro-, sulfonium salt, and two spirocyclic iodonium ylide (SCIDY) precursors bearing a cyclopentyl (SPI5) and a new adamantyl (SPIAd) auxiliary. The chloro- and nitro-precursors resulted in a low radiochemical yield (<10% RCY), whereas both SCIDY precursors and the sulfonium salt precursor produced [18F]FPEB in the highest RCYs of 25% and 36%, respectively. Preliminary PET/CT imaging studies with [18F]FPEB were conducted in a transgenic model of Alzheimer’s Disease (AD) using B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J (APP/PS1) mice, and data were compared with age-matched wild-type (WT) B6C3F1/J control mice. In APP/PS1 mice, whole brain distribution at 5 min post-injection showed a slightly higher uptake (SUV = 4.8 ± 0.4) than in age-matched controls (SUV = 4.0 ± 0.2). Further studies to explore mGluR5 as an early biomarker for AD are underway.


Radiosyntheses of [ 18 F]FPEB with Five Different Precursors
The chloro-precursor (1) was synthesized following a literature procedure, with the modification that conventional heating was used to drive the radiofluorination instead of the previously reported microwave conditions [9]. In our hands, a maximum 3% RCC was observed by manual synthesis at 200 ℃ from a 10 min reaction. In light of the low radiofluorination conversion, this precursor was not considered for automated radiosynthesis ( Table 2, Entry I). While manual radiosynthesis of [ 18 F]FPEB via the commercially available nitro-precursor (2) showed an RCC based on radio-HPLC analysis of 33% (n = 3) at 150 °C for a reaction time of 5 min, upon automation and isolation, the highest RCY was only 4% ( Table 2, Entry II). This low RCY from the nitro-precursor is consistent with other reported production yields [8,10,18,19]. Losses experienced when translated to an automated synthesis unit may be attributed to transfer to tubing and reaction vessels. The low yielding reaction, together with the presence of radiochemical and UV active impurities, observed by HPLC, led us to abandon the optimization of [ 18 F]FPEB radiosynthesis using the nitro-precursor and explore alternative precursors.

Radiosyntheses of [ 18 F]FPEB with Five Different Precursors
The chloro-precursor (1) was synthesized following a literature procedure, with the modification that conventional heating was used to drive the radiofluorination instead of the previously reported microwave conditions [9]. In our hands, a maximum 3% RCC was observed by manual synthesis at 200 ℃ from a 10 min reaction. In light of the low radiofluorination conversion, this precursor was not considered for automated radiosynthesis ( Table 2, Entry I). While manual radiosynthesis of [ 18 F]FPEB via the commercially available nitro-precursor (2) showed an RCC based on radio-HPLC analysis of 33% (n = 3) at 150 °C for a reaction time of 5 min, upon automation and isolation, the highest RCY was only 4% (Table 2, Entry II). This low RCY from the nitro-precursor is consistent with other reported production yields [8,10,18,19]. Losses experienced when translated to an automated synthesis unit may be attributed to transfer to tubing and reaction vessels. The low yielding reaction, together with the presence of radiochemical and UV active impurities, observed by HPLC, led us to abandon the optimization of [ 18 F]FPEB radiosynthesis using the nitro-precursor and explore alternative precursors.

Radiosyntheses of [ 18 F]FPEB with Five Different Precursors
The chloro-precursor (1) was synthesized following a literature procedure, with the modification that conventional heating was used to drive the radiofluorination instead of the previously reported microwave conditions [9]. In our hands, a maximum 3% RCC was observed by manual synthesis at 200 ℃ from a 10 min reaction. In light of the low radiofluorination conversion, this precursor was not considered for automated radiosynthesis ( Table 2, Entry I). While manual radiosynthesis of [ 18 F]FPEB via the commercially available nitro-precursor (2) showed an RCC based on radio-HPLC analysis of 33% (n = 3) at 150 °C for a reaction time of 5 min, upon automation and isolation, the highest RCY was only 4% (Table 2, Entry II). This low RCY from the nitro-precursor is consistent with other reported production yields [8,10,18,19]. Losses experienced when translated to an automated synthesis unit may be attributed to transfer to tubing and reaction vessels. The low yielding reaction, together with the presence of radiochemical and UV active impurities, observed by HPLC, led us to abandon the optimization of [ 18 F]FPEB radiosynthesis using the nitro-precursor and explore alternative precursors.

Radiosyntheses of [ 18 F]FPEB with Five Different Precursors
The chloro-precursor (1) was synthesized following a literature procedure, with the modification that conventional heating was used to drive the radiofluorination instead of the previously reported microwave conditions [9]. In our hands, a maximum 3% RCC was observed by manual synthesis at 200 ℃ from a 10 min reaction. In light of the low radiofluorination conversion, this precursor was not considered for automated radiosynthesis ( Table 2, Entry I). While manual radiosynthesis of [ 18 F]FPEB via the commercially available nitro-precursor (2) showed an RCC based on radio-HPLC analysis of 33% (n = 3) at 150 °C for a reaction time of 5 min, upon automation and isolation, the highest RCY was only 4% ( Table 2, Entry II). This low RCY from the nitro-precursor is consistent with other reported production yields [8,10,18,19]. Losses experienced when translated to an automated synthesis unit may be attributed to transfer to tubing and reaction vessels. The low yielding reaction, together with the presence of radiochemical and UV active impurities, observed by HPLC, led us to abandon the optimization of [ 18 F]FPEB radiosynthesis using the nitro-precursor and explore alternative precursors.

Radiosyntheses of [ 18 F]FPEB with Five Different Precursors
The chloro-precursor (1) was synthesized following a literature procedure, with the modification that conventional heating was used to drive the radiofluorination instead of the previously reported microwave conditions [9]. In our hands, a maximum 3% RCC was observed by manual synthesis at 200 ℃ from a 10 min reaction. In light of the low radiofluorination conversion, this precursor was not considered for automated radiosynthesis ( Table 2, Entry I). While manual radiosynthesis of [ 18 F]FPEB via the commercially available nitro-precursor (2) showed an RCC based on radio-HPLC analysis of 33% (n = 3) at 150 °C for a reaction time of 5 min, upon automation and isolation, the highest RCY was only 4% ( Table 2, Entry II). This low RCY from the nitro-precursor is consistent with other reported production yields [8,10,18,19]. Losses experienced when translated to an automated synthesis unit may be attributed to transfer to tubing and reaction vessels. The low yielding reaction, together with the presence of radiochemical and UV active impurities, observed by HPLC, led us to abandon the optimization of [ 18 F]FPEB radiosynthesis using the nitro-precursor and explore alternative precursors.

Radiosyntheses of [ 18 F]FPEB with Five Different Precursors
The chloro-precursor (1) was synthesized following a literature procedure, with the modification that conventional heating was used to drive the radiofluorination instead of the previously reported microwave conditions [9]. In our hands, a maximum 3% RCC was observed by manual synthesis at 200 ℃ from a 10 min reaction. In light of the low radiofluorination conversion, this precursor was not considered for automated radiosynthesis (  (2) showed an RCC based on radio-HPLC analysis of 33% (n = 3) at 150 °C for a reaction time of 5 min, upon automation and isolation, the highest RCY was only 4% ( Table 2, Entry II). This low RCY from the nitro-precursor is consistent with other reported production yields [8,10,18,19]. Losses experienced when translated to an automated synthesis unit may be attributed to transfer to tubing and reaction vessels. The low yielding reaction, together with the presence of radiochemical and UV active impurities, observed by HPLC, led us to abandon the optimization of [ 18 F]FPEB radiosynthesis using the nitro-precursor and explore alternative precursors.

Radiosyntheses of [ 18 F]FPEB with Five Different Precursors
The chloro-precursor (1) was synthesized following a literature procedure, with the modification that conventional heating was used to drive the radiofluorination instead of the previously reported microwave conditions [9]. In our hands, a maximum 3% RCC was observed by manual synthesis at 200 ℃ from a 10 min reaction. In light of the low radiofluorination conversion, this precursor was not considered for automated radiosynthesis (  (2) showed an RCC based on radio-HPLC analysis of 33% (n = 3) at 150 °C for a reaction time of 5 min, upon automation and isolation, the highest RCY was only 4% (Table 2, Entry II). This low RCY from the nitro-precursor is consistent with other reported production yields [8,10,18,19]. Losses experienced when translated to an automated synthesis unit may be attributed to transfer to tubing and reaction vessels. The low yielding reaction, together with the presence of radiochemical and UV active impurities, observed by HPLC, led us to abandon the optimization of [ 18 F]FPEB radiosynthesis using the nitro-precursor and explore alternative precursors.
We recently showed that using the SCIDY chemistry with a cyclopentyl auxiliary (SPI5) (5) as the precursor for [ 18 F]FPEB production led to a five-fold increase in RCY and a three-fold increase in molar activity (Am) compared to the nitro-precursor. The SCIDY precursor enabled the displacement reaction with [ 18 F]fluoride to be conducted at milder conditions (lower temperatures and shorter times), thereby minimizing the formation of radiochemical impurities. [  Manual [16] * not isolated.

Radiosyntheses of [ 18 F]FPEB with Five Different Precursors
The chloro-precursor (1) was synthesized following a literature procedure, with the modification that conventional heating was used to drive the radiofluorination instead of the previously reported microwave conditions [9]. In our hands, a maximum 3% RCC was observed by manual synthesis at 200°C from a 10 min reaction. In light of the low radiofluorination conversion, this precursor was not considered for automated radiosynthesis (  (2) showed an RCC based on radio-HPLC analysis of 33% (n = 3) at 150 • C for a reaction time of 5 min, upon automation and isolation, the highest RCY was only 4% (Table 2, Entry II). This low RCY from the nitro-precursor is consistent with other reported production yields [8,10,18,19]. Losses experienced when translated to an automated synthesis unit may be attributed to transfer to tubing and reaction vessels. The low yielding reaction, together with the presence of radiochemical and UV active impurities, observed by HPLC, led us to abandon the optimization of [ 18 F]FPEB radiosynthesis using the nitro-precursor and explore alternative precursors.
We recently showed that using the SCIDY chemistry with a cyclopentyl auxiliary (SPI5) (5) as the precursor for [ 18 F]FPEB production led to a five-fold increase in RCY and a three-fold increase in molar activity (A m ) compared to the nitro-precursor. The SCIDY precursor enabled the displacement reaction with [ 18 F]fluoride to be conducted at milder conditions (lower temperatures and shorter times), thereby minimizing the formation of radiochemical impurities. [  time) which resulted in a RCY of 25% ± 2%, with a molar activity (Am) of = 37 ± 13 GBq/µmol (n = 3), consistent with our previous reports [13,15]. A recently reported precursor (6) based on a sulfonium salt was also applied to the manual synthesis of [ 18 F]FPEB and gave a reported 55% RCY [16]. Inspired by this report, we adapted the radiochemistry precursor and methodology for automated radiofluorination with the GE TRACERlab™ FX2 N synthesis module. The reaction was conducted at varying temperatures (80 or 100 ℃) for 5 min and with varying bases (Et4NHCO3 or KHCO3/K222) to establish optimal temperature and base conditions, as summarized in Table 2, with the literature synthesis providing the highest yield [16]. Using [K222][ 18 F] and the milder base (KHCO3) at 80 °C for 5 min (Table 2, Entry V), the sulfonium salt precursor produced [ 18 F]FPEB with a RCY of 36% ± 6% and Am = 77 ± 35 GBq/µmol (n = 3). When considering which chemical route should be used to produce [ 18 F]FPEB for clinical research, many factors will impact this choice, including precursor availability, radiochemical yield, molar activity, ease of automation and purification, etc. Although the nitro-precursor is currently the only commercially available compound for [ 18 F]FPEB production, the resulting yields are much lower than the SCIDY and sulfonium salt precursors. The SPI5 auxiliary and the sulfonium salt precursors appear to the be the best suited for routine radiopharmaceutical production of [ 18 F]FPEB.

Small Animal PET/CT Imaging
mGluR5 has emerged as an imaging target in AD pathogenesis. It has been demonstrated that soluble oligomeric amyloid-β (Aβo) induces an accumulation and over-stabilization of mGluR5, and that Aβo up-regulates mGluR5, leading to an abnormal increase in the release of intracellular Ca 2+  [17,20]. The SPIAd precursor (compound 7) also resulted in a 24% RCY for [ 18 F]FPEB, and is similar to that of the SPI5 precursor ( Table 2, Entry IV). Our initial semi-preparative HPLC purification conditions did not adequately separate the adamantyl precursor from [ 18 F]FPEB, and given the equivalent RCY between the two different SCIDY auxiliaries, optimization of the SPIAd precursor reaction and/or HPLC conditions were not pursued (Supplementary Materials).
A recently reported precursor (6) based on a sulfonium salt was also applied to the manual synthesis of [ 18 F]FPEB and gave a reported 55% RCY [16]. Inspired by this report, we adapted the radiochemistry precursor and methodology for automated radiofluorination with the GE TRACERlab™ FX2 N synthesis module. The reaction was conducted at varying temperatures (80 or 100 • C) for 5 min and with varying bases (Et 4 NHCO 3 or KHCO 3 /K 222 ) to establish optimal temperature and base conditions, as summarized in Table 2, with the literature synthesis providing the highest yield [16]. Using [K 222 ][ 18 F] and the milder base (KHCO 3 ) at 80 • C for 5 min (Table 2, Entry V), the sulfonium salt precursor produced [ 18 F]FPEB with a RCY of 36% ± 6% and Am = 77 ± 35 GBq/µmol (n = 3).
When considering which chemical route should be used to produce [ 18 F]FPEB for clinical research, many factors will impact this choice, including precursor availability, radiochemical yield, molar activity, ease of automation and purification, etc. Although the nitro-precursor is currently the only commercially available compound for [ 18 F]FPEB production, the resulting yields are much lower than the SCIDY and sulfonium salt precursors. The SPI5 auxiliary and the sulfonium salt precursors appear to the be the best suited for routine radiopharmaceutical production of [ 18 F]FPEB.

Small
Animal PET/CT Imaging mGluR5 has emerged as an imaging target in AD pathogenesis. It has been demonstrated that soluble oligomeric amyloid-β (Aβo) induces an accumulation and over-stabilization of mGluR5, and that Aβo up-regulates mGluR5, leading to an abnormal increase in the release of intracellular Ca 2+ [6,[21][22][23]. Preliminary PET imaging data with [ 18 F]FPEB showed increased brain uptake in the transgenic model of AD versus the age-matched controls (10 month data shown in Figure 1). Dynamic PET/CT imaging was carried out to investigate the difference in [ 18 F]FPEB binding between an established preclinical model of AD using 10 month old transgenic APP/PS1 mice and their Molecules 2020, 25, 982 5 of 9 age-matched wild-type (WT) controls. Axial, coronal, and sagittal images of the murine brains acquired at 20 min post-injection of [ 18 F]FPEB are presented in Figure 1A. A marked increased uptake of radiotracer binding was observed in the brains of the transgenic mice compared with WT controls. The time-activity curves revealed a similar initial peak uptake and brain penetration for both groups of mice ( Figure 1B). One minute after the time of injection (TOI), maximum uptake was observed in both genotypes, (SUV > 6; whole brain VOI). By 10 min post-injection, a modest higher [ 18 F]FPEB retention was observed in the brain of transgenic mice (SUV = 4.8) versus WT controls (SUV = 4.0), and as the tracer cleared from normal tissues, a significant difference (p < 0.05) was apparent at <5 min post-injection. A comparison of the area under the curve (AUC) analysis ( Figure 1C) over time for the respected genotypes underscores the trends observed in the TACs.
Molecules 2019, 24, x FOR PEER REVIEW 2 of 3 [6,[21][22][23]. Preliminary PET imaging data with [ 18 F]FPEB showed increased brain uptake in the transgenic model of AD versus the age-matched controls (10 month data shown in Figure 1). Dynamic PET/CT imaging was carried out to investigate the difference in [ 18 F]FPEB binding between an established preclinical model of AD using 10 month old transgenic APP/PS1 mice and their agematched wild-type (WT) controls. Axial, coronal, and sagittal images of the murine brains acquired at 20 min post-injection of [ 18 F]FPEB are presented in Figure 1A. A marked increased uptake of radiotracer binding was observed in the brains of the transgenic mice compared with WT controls. The time-activity curves revealed a similar initial peak uptake and brain penetration for both groups of mice ( Figure 1B). One minute after the time of injection (TOI), maximum uptake was observed in both genotypes, (SUV > 6; whole brain VOI). By 10 min post-injection, a modest higher [ 18 F]FPEB retention was observed in the brain of transgenic mice (SUV = 4.8) versus WT controls (SUV = 4.0), and as the tracer cleared from normal tissues, a significant difference (p < 0.05) was apparent at <5 min post-injection. A comparison of the area under the curve (AUC) analysis ( Figure 1C) over time for the respected genotypes underscores the trends observed in the TACs. In a pilot study, we found that patients with early mild cognitive impairment had an increased brain uptake of [ 18 F]FPEB, and that radiotracer uptake in the brain was reflective of increased mGluR5 density [10]. This observation supports the hypothesis that mGluR5 may be implicated in the early stages of AD pathogenesis [24]. Consistent with this hypothesis and clinical research indication, PET/CT imaging studies of [ 18 F]FPEB uptake in a transgenic mouse model of AD also showed an increased radiotracer uptake and retention in the brain of the APP/PS1 mice, compared with wildtype controls. This preliminary work provides support that mGluR5 levels measured by [ 18 F]FPEB are potentially useful as an early biomarker of AD. Further [ 18 F]FPEB imaging studies and biological evaluations are underway including regional analysis of imaging data as well as ex vivo biodistribution and autoradiography studies to evaluate this functional link between mGluR5 and AD. In a pilot study, we found that patients with early mild cognitive impairment had an increased brain uptake of [ 18 F]FPEB, and that radiotracer uptake in the brain was reflective of increased mGluR5 density [10]. This observation supports the hypothesis that mGluR5 may be implicated in the early stages of AD pathogenesis [24]. Consistent with this hypothesis and clinical research indication, PET/CT imaging studies of [ 18 F]FPEB uptake in a transgenic mouse model of AD also showed an increased radiotracer uptake and retention in the brain of the APP/PS1 mice, compared with wild-type controls. This preliminary work provides support that mGluR5 levels measured by [ 18 F]FPEB are potentially useful as an early biomarker of AD. Further [ 18 F]FPEB imaging studies and biological evaluations are underway including regional analysis of imaging data as well as ex vivo biodistribution and autoradiography studies to evaluate this functional link between mGluR5 and AD.

Materials and General Methods
Unless otherwise stated, all reagents were obtained from commercially available sources and used without further purification. The identification of all radiochemical products was determined by HPLC co-elution with an authentic non-radioactive standard. All RCC and RCY values are reported as decay corrected, relative to starting [ 18  Preparation of the chloro-precursor was performed as previously reported [9]. The nitro-precursor (Lot No.: 20190401) and FPEB standard (Lot No.: 20130101) were purchased from ABX. Synthesis of the sulfonium salt precursor and radiolabeling were performed as previously reported [16]. For manual labeling, azeotropically dried potassium cryptand [ 18 F]fluoride was dissolved in DMSO (3.0 mL), while 400 µL aliquots (typically 3-5 mCi) were used per reaction to dissolve the precursor in a 1 dram vial. The reaction was heated at 150 • C for 5 min, then quenched with water and cooled for 3 min. Product identity and radiochemical conversion were determined as the ratio of free [ 18 F]fluoride to [ 18 F]FPEB as integrated by radio-HPLC. The manual radiolabeling of the chloro-(1) and nitro-(2) precursors was performed as previously reported, with slight modification to the concentration and reaction time. One milligram of 2 was dissolved in 0.4 mL [ 18 F]fluoride in DMSO for 5 min instead of 1.5 mL for 15 min [8,18], and the chloro-reaction was conducted at high temperatures instead of under microwave conditions [9]. For automated radiosyntheses, a GE TRACERlab™ FX2 N module with 2 and the SPI5 auxiliary SCIDY precursor (5) were performed as previously reported [13,15]. Flash chromatography was performed on a Biotage Isolera One automated flash purification system. Biotage SNAP KP-Sil 50 g cartridges (45-60 micron) were used with a flow rate of 50 mL/min for gradient solvent systems. Fractions were monitored and collected by UV absorbance using the internal UV detector set at 254 and 280 nm.

SCIDY-SPIAd Auxilary Synthesis and Characterization
The titled compound was prepared using a modified literature procedure [15]. Trifluoroacetic acid (0.9 mL) was added to a solution of IPEB (120 mg, 0.36 mmol) in chloroform (0.12 mL). Oxone (179 mg, 0.58 mmol) was added and the reaction mixture was stirred for 3 h, until full conversion of starting materials was determined by TLC (SiO 2 coated on polyethylene, 250 µm, with 100% EtOAc). Volatile contents were then removed by rotary evaporation. The round bottom flask was covered in foil and further dried under high vacuum for 5 h. The dried residue was suspended in ethanol (1.5 mL) and (1r,3r,5r,7r)-spiro[adamantane-2,2 -[1,3]dioxane]-4 ,6 -dione (67 mg, 0.54 mmol). SPIAd was added followed by 10% Na 2 CO 3 (aq) (w/v, 1.5 mL, 0.33 M solution) in~0.2 mL aliquots until the pH of the reaction mixture was equal to pH 10. The reaction mixture was stirred for 5 h until full conversion to the iodonium ylide was determined by TLC (SiO 2 coated on polyethylene, 250 µm, with 10% EtOH in EtOAc, (1:9 mL v/v)). The reaction mixture was then diluted with water and extracted with chloroform. The chloroform extracts were combined and washed with water (4 × 10 mL) and brine (1 × 10 mL). The organic layer was dried with anhydrous MgSO 4 , filtered, and concentrated. The final compound was purified by flash chromatography using a gradient 60% EtOAc in hexanes to 100% EtOAc to 5% methanol in EtOAc. Compound 7 (56 mg, 0.11 mmol) was isolated as an off-white powder with a 41% yield. determine the maximum and mean SUV radiotracer uptake in various tissues. Time-activity curves (TACs) were generated from the ROI analysis on dynamic PET/CT data using 20 s frames. CT images were recorded using an X-ray current of 300 µA, 360 projections, and an image size of 63.8 mm × 63.8 mm × 46.0 mm. Data were acquired using the Vista CT 4.11 Build 701 software, and reconstructed images were analyzed by using ASIPro VM TM software (Concorde Microsystems, Siemens Preclinical Solutions, LLC, Knoxville, TN, USA) and VivoQuant ® 1.23 (InviCRO, LLC, Boston, MA, USA).

Data Analysis and Statistics
Data and statistical analyses were performed using GraphPad Prism 5.01 (GraphPad Software, Inc., La Jolla, CA, USA) and Microsoft Excel spreadsheets. Differences at the 95% confidence level (p < 0.05) were considered to be statistically significant.