Skip to main content

Automated radiosynthesis of two 18F-labeled tracers containing 3-fluoro-2-hydroxypropyl moiety, [18F]FMISO and [18F]PM-PBB3, via [18F]epifluorohydrin

Abstract

Background

[18F]Fluoromisonidazole ([18F]FMISO) and 1-[18F]fluoro-3-((2-((1E,3E)-4-(6-(methylamino)pyridine-3-yl)buta-1,3-dien-1-yl)benzo[d]thiazol-6-yl)oxy)propan-2-ol ([18F]PM-PBB3 or [18F]APN-1607) are clinically used radiotracers for imaging hypoxia and tau pathology, respectively. Both radiotracers were produced by direct 18F-fluorination using the corresponding tosylate precursors 1 or 2 and [18F]F, followed by the removal of protecting groups. In this study, we synthesized [18F]FMISO and [18F]PM-PBB3 by 18F-fluoroalkylation using [18F]epifluorohydrin ([18F]5) for clinical applications.

Results

First, [18F]5 was synthesized by the reaction of 1,2-epoxypropyl tosylate (8) with [18F]F and was purified by distillation. Subsequently, [18F]5 was reacted with 2-nitroimidazole (6) or PBB3 (7) as a precursor for 18F-labeling, and each reaction mixture was purified by preparative high-performance liquid chromatography and formulated to obtain the [18F]FMISO or [18F]PM-PBB3 injection. All synthetic sequences were performed using an automated 18F-labeling synthesizer. The obtained [18F]FMISO showed sufficient radioactivity (0.83 ± 0.20 GBq at the end of synthesis (EOS); n = 8) with appropriate radiochemical yield based on [18F]F (26 ± 7.5 % at EOS, decay-corrected; n = 8). The obtained [18F]PM-PBB3 also showed sufficient radioactivity (0.79 ± 0.10 GBq at EOS; n = 11) with appropriate radiochemical yield based on [18F]F (16 ± 3.2 % at EOS, decay-corrected; n = 11).

Conclusions

Both [18F]FMISO and [18F]PM-PBB3 injections were successfully synthesized with sufficient radioactivity by 18F-fluoroalkylation using [18F]5.

Background

Fluorine-18 (T1/2 = 109.8 min) is indispensable for the development of positron emission tomography (PET) tracers because its decay characteristic is better than that of carbon-11 (T1/2 = 20.1 min). The direct 18F-fluorination using a tosylate or triflate precursor and [18F]F is a widely used method for the introduction of fluorine-18 into target molecules to afford a large number of 18F-labeled PET tracers (Cole et al. 2014; Deng et al. 2019; Miller et al. 2008). In addition, 18F-fluoroalkylation is also a useful tool for inserting fluorine-18 into target molecules containing nucleophilic hydroxyl and amino functional groups (Zhang and Suzuki 2007). 18F-Fluoroalkylation has some advantages over direct 18F-fluorination. For example, 18F-fluoroalkylation applies more accessible and available phenols, carboxylic acids, amines, and amides as precursors for 18F-labeling (Iwata et al. 2002; Wilson et al. 1995; Zhang and Suzuki 2007). We have synthesized 18F-fluoroalkyl agents, such as [18F]fluoro-methyl, ethyl, and propyl bromide ([18F]F(CH2)nBr, n = 1–3) (Yanamoto et al. 2009; Yui et al. 2010; Zhang et al. 2002, 2003, 2004; Zhang and Suzuki 2007), deuterium-substituted [18F]fluoromethyl bromide ([18F]FCD2Br), and its triflate ([18F]FCD2OTf) using an automated 18F-labeling synthesizer (Arakawa et al. 2008; Mori et al. 2019). Using these 18F-fluoroalkyl agents, we synthesized dozens of 18F-fluoroalkylated tracers starting from the precursors of phenols, carboxylic acids, amines, and amides for PET imaging of receptors, enzymes, and transporters in the brain (Zhang and Suzuki 2007). Among these PET tracers, [18F]FEDAA1106 (Fujimura et al. 2006), [18F]FE-SPARQ (Haneda et al. 2007), [18F]FMeNER-d2 (Arakawa et al. 2008), [18F]FEPE2I (Sasaki et al. 2012), and [18F]FEDAC (Chung et al. 2018; Xie et al. 2012) have been synthesized for clinical applications in our PET center.

The 18F-3-fluoro-2-hydroxypropyl (18F-FHP) moiety was used instead of the aforementioned conventional 18F-fluoroalkyl moieties. Many PET tracers containing the 18F-FHP moiety have been developed, some of which have been used in clinical studies, such as [18F]FMISO (Bruehlmeier et al. 2004; Eschmann et al. 2005), [18F]THK-5351 (Harada et al. 2016; Tago et al. 2016), [18F]FC1195S (Byun et al. 2017; Lee et al. 2016; Yang et al. 2016), [18F]SMBT-1 (Harada et al. 2021), and [18F]PM-PBB3 (Tagai et al. 2021; Kawamura et al. 2021) (Fig. 1). Among these PET tracers, [18F]THK-5351 (Fig. 1), which contains the FHP moiety, showed improved in vivo metabolic stability compared with its fluoropropyl analog (Tago et al. 2016).

Fig. 1
figure 1

Chemical structures of PET tracers containing (3-[18F]fluoro-2-hydroxy)propyl ([18F]FHP) moiety used in clinical applications. [18F]FMISO (Bruehlmeier et al. 2004; Eschmann et al. 2005); [18F]THK-5351 (Harada et al. 2016; Tago et al. 2016); [18F]FC1195S (Byun et al. 2017; Lee et al. 2016; Yang et al. 2016); [18F]SMBT-1 (Harada et al. 2021); [18F]PM-PBB3 (Tagai et al. 2021)

To date, many 18F-labeled tracers containing 18F-fluoroalkyl moieties have been developed. To synthesize these PET tracers, direct 18F-fluorination of the corresponding tosylate or triflate precursor with [18F]F is a conventional method. Among these, [18F]FMISO as a PET imaging agent for tumor hypoxia (Oh et al. 2005; Tang et al. 2005), and [18F]PM-PBB3 as a PET imaging agent for tau pathology (Kawamura et al. 2021) have been prepared by direct 18F-fluorination using tosylate precursors and [18F]F, followed by the removal of the protecting group. The direct 18F-fluorination was achieved within the same reaction vessel using an automated synthesizer. Moreover, the one-step radiolabeling technique for 18F-labeled tracers could be readily transferred to other PET centers for multisite studies using the same study protocol (Kawamura et al. 2016, 2021; Mori et al. 2017). In fact, automated radiosynthesis of [18F]PM-PBB3 by direct 18F-fluorination has been transferred to a dozen PET centers in Japan, China, Taiwan, and the USA (Hsu et al. 2020; Su et al. 2020; Weng et al. 2020). As for the limitation of these direct 18F-fluorination, it is noted that tosylated precursors should be synthesized in at least two steps. [18F]FMISO was synthesized using [18F]epfluorohydrin ([18F]5), as described previously (Grierson et al. 1989; Kämäräinen et al. 2004; McCarthy et al. 1993). In those papers, fully automated radiosynthesis procedures of [18F]FMISO via [18F]5 using an 18F-labeling synthesizer have not been reported.

In this study, to determine an effective synthetic route for [18F]FMISO and [18F]PM-PBB3 with sufficient radioactivity and high quality for clinical applications, we synthesized the two PET tracers using [18F]5 as an 18F-labeling agent by the reaction of easily accessible 2-nitroimidazole (6, Fig. 2) or PBB3 (a phenol precursor; 7, Fig. 3) using an 18F-labeling synthesizer equipped with a fully automated system. Furthermore, we compared the synthetic results of 18F-fluoroalkylation using [18F]5 and 18F-fluorination using [18F]F to evaluate their relative merits.

Fig. 2
figure 2

Radiosynthesis of [18F]FMISO by 18F-fluorination using [18F]F and 18F-fluoroalkylation using [18F]epifluorohydrin ([18F]5). In this study, after the [18F]F solution was dried, a solution of 8 (3.5 mg) in o-dichlorobenzene (0.15 mL) was added to a reaction vial containing dry [18F]F. The resulting [18F]5 was distilled under an atmosphere of nitrogen, and transferred to another reaction vial containing 6 (2 mg) and 1 mol/L NaOH (18 µL) in anhydrous DMF (0.25 mL) maintained at -15 °C. After 2 min of trapping of [18F]5, the reaction mixture was heated at 150 °C for 20 min to obtain [18F]FMISO

Fig. 3
figure 3

Radiosynthesis of [18F]PM-PBB3 by 18F-fluorination with [18F]F and 18F-fluoroalkylation using [18F]epifluorohydrin ([18F]5). In this study, after the [18F]F solution was dried, a solution of 8 (3.5 mg) in o-dichlorobenzene (0.15 mL) was added to the reaction vial containing the dry [18F]F. The resulting [18F]5 was distilled from the reaction vial and was transferred to another reaction vial containing 7 (1 mg) and 1 mol/L NaOH (3.5 µL) in anhydrous DMF (0.25 mL) maintained at -15 °C. After 2 min of trapping of [18F]5, the reaction mixture was heated at 130 °C for 20 min to obtain [18F]PM-PBB3

Methods

General

1 H-1-(3-Fluoro-2-hydroxypropyl)-2-nitroimidazole (FMISO, Fig. 1) and 2-nitroimidazole (6, Fig. 2) were purchased from ABX (Radeberg, Germany). 1-Fluoro-3-((2-((1E,3E)-4-(6-(methylamino)pyridine-3-yl)buta-1,3-dien-1-yl)benzo[d]thiazol-6-yl)oxy)propan-2-ol (PM-PBB3, Fig. 1), and 2-((1E,3E)-4-(6-(methylamino)pyridin-3-yl)buta-1,3-dienyl)benzo[d]thiazol-6-ol (PBB3; 7, Fig. 3) (Hashimoto et al. 2014; Maruyama et al. 2013) were provided by Shanghai ChemPartner (Shanghai, China). All chemical reagents and organic solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA), Fujifilm Wako Pure Chemical Co. (Osaka, Japan), and Nacalai Tesque (Kyoto, Japan), and were used without any further purification. Fluorine-18 was produced by the 18O(p, n)18F nuclear reaction using a CYPRIS HM-18 cyclotron (Sumitomo Heavy Industry, Tokyo, Japan). A dose calibrator (IGC-3R Curiemeter; Aloka, Tokyo, Japan) was used for all radioactivity measurements, unless otherwise stated. An automated multi-purpose synthesizer developed in-house was used for all the radiosynthetic runs in this study (Supplemental information: Fig. S1, Fukumura et al. 2007). Preparative high-performance liquid chromatography (HPLC) was performed using a JASCO HPLC system (PU-2080 pump and UV-2075 detector; JASCO, Tokyo, Japan) equipped with a radioactivity detector (Ohyo Koken Kogyo, Tokyo, Japan). All radiochemical yields were decay-corrected to the end of synthesis. Fluorine-18, as [18F]F, was produced as described previously (Fujinaga et al. 2018).

Automated radiosynthesis of [18F]FMISO using [18F]5

After the [18F]F solution (5.2 ± 0.20 GBq, n = 8) was dried, a solution of epoxypropyl tosylate (8, 3.5 mg) in o-dichlorobenzene (0.15 mL) was added to the reaction vial containing dry [18F]F at 130 °C. The resulting [18F]5 was distilled from the reaction vial under N2 flow at 30 mL/min and was transferred to another reaction vial containing precursor 6 (2 mg) and 1 mol/L sodium hydroxide solution (NaOH, 18 µL) in anhydrous N,N-dimethylformamide (DMF, 0.25 mL) maintained at − 15 °C. After 2 min of trapping of [18F]5, the reaction mixture was heated at 150 °C for 20 min, and then was diluted with the preparative HPLC eluent (0.5 mL). The solution mixture was transferred to the injector for preparative HPLC, as described in the general section. The HPLC conditions were as follows: XBridge C18 column (5 μm, 10 mm i.d. × 250 mm length; Waters), a mixture of ethanol and water (2:98, vol./vol.) as the mobile phase, 5.0 mL/min flow rate, and UV detection at 325 nm. The retention time of [18F]FMISO was approximately 12 min. The HPLC fraction of [18F]FMISO was collected in a flask containing polysorbate 80 (75 µL) in ethanol (0.3 mL), and ascorbic acid for injection (25 mg/0.1 mL water) was added before radiosynthesis. The solution was subsequently evaporated to dryness, and the residue was dissolved in physiological saline (3–10 mL). The resulting solution was passed through a Millex-GV filter (Millipore) to obtain [18F]FMISO as an injectable solution.

The radiochemical purity of [18F]FMISO was determined using analytical HPLC under the following conditions: XBridge C18 column (5 μm, 4.6 mm i.d. × 150 mm length; Waters), a mixture of 90 % acetonitrile solution and 50 mM ammonium phosphate buffer (pH 9.3) (7:93, vol./vol.) as the mobile phase, 1.0 mL/min flow rate, and UV detection at 325 nm. The retention time of [18F]FMISO was 5.6 min. The identity of [18F]FMISO was confirmed by co-injecting it with authentic unlabeled FMISO. The molar activity of [18F]FMISO was measured using the same analytical HPLC system. The mass (µmol) of FMISO with a known radioactivity (GBq) was determined using analytical HPLC by comparing the UV absorbance at 325 nm of [18F]FMISO with that of known concentrations of unlabeled FMISO.

Automated radiosynthesis of [18F]PM-PBB3 using [18F]5

After the [18F]F solution (7.4 ± 0.20 GBq, n = 11) was dried, a solution of epoxypropyl tosylate (8, 3.5 mg) in o-dichlorobenzene (0.15 mL) was added automatically to the reaction vial containing the dry [18F]F at 130 °C. The resulting [18F]5 was distilled from the reaction vial under N2 flow at 30 mL/min and was transferred to another reaction vial containing precursor 7 (1 mg) and 1 mol/L NaOH (3.5 µL) in anhydrous DMF (0.25 mL) maintained at − 15 °C. After 2 min of trapping of [18F]5, the reaction mixture was heated at 130 °C for 20 min, and then was diluted with the preparative HPLC eluent (0.5 mL). The solution was transferred to the injector for preparative HPLC, as described in the general section. The HPLC conditions were as follows: Capcell Pak C18 column (5 μm, 10 mm i.d. × 250 mm length; Shiseido, Tokyo, Japan), a mixture of acetonitrile, water, and triethylamine (40:60:0.1, vol./vol./vol.) as the mobile phase, 5.0 mL/min flow rate, and UV detection at 365 nm. The retention time of [18F]PM-PBB3 was 14.9 min. The HPLC fraction of [18F]PM-PBB3 was collected in a flask containing polysorbate 80 (75 µL) in ethanol (0.3 mL) and ascorbic acid for injection (25 mg/0.1 mL water) was added before radiosynthesis. The solution was subsequently evaporated to dryness, and the residue was dissolved in physiological saline (3–10 mL). The solution of [18F]PM-PBB3 was passed through a Millex-GV filter to obtain [18F]PM-PBB3 as an injectable solution. The preparative HPLC and formulation were performed under UV-cut light (< 500 nm wavelength cutoff, ECOHiLUX HES-YF; Iris Oyama, Sendai, Japan) to prevent the photoisomerization of [18F]PM-PBB3, because [18F]PM-PBB3 underwent rapid photoisomerization upon exposure to fluorescent light (Kawamura et al. 2021).

The radiochemical purity of [18F]PM-PBB3 was determined by analytical HPLC under the following conditions: Atlantis T3 column (5 μm, 4.6 mm i.d. × 150 mm length; Waters), a mixture of acetonitrile and 50 mM ammonium acetate (pH 6.5) (40:60, vol./vol.) as the mobile phase, 1.0 mL/min flow rate, and UV detection at 365 nm. The retention time of [18F]PM-PBB3 was 12 min. The identity of [18F]PM-PBB3 was confirmed by co-injecting it with authentic unlabeled PM-PBB3. The molar activity of [18F]PM-PBB3 was measured using the same analytical HPLC system. The mass (µmol) of [18F]PM-PBB3 with a known radioactivity (GBq) was determined by comparing the UV absorbance at 365 nm of PM-PBB3 with that of known concentrations of unlabeled PM-PBB3. All of the above analytical processes were conducted in the absence of fluorescent light to prevent the photoisomerization of [18F]PM-PBB3.

Results

Automated radiosynthesis of [18F]FMISO using [18F]5

We synthesized [18F]FMISO under various reaction conditions for the 18F-fluoroalkylation of 2-nitroimidazole precursor 6 and [18F]5 using an automated 18F-labeling synthesizer. With an increase in the amount of 6 from 0.5 to 4 mg, the radiochemical yield of [18F]FMISO gradually increased to 36 % from 0.5 to 2 mg, and marginally increased up to 42 % from 2 to 4 mg [Fig. 4(A)]. In addition, increasing the reaction temperature from 90 to 150 °C increased the radiochemical yield of [18F]FMISO by up to 40 % [Fig. 4(B)]. Furthermore, the radiochemical yield obtained by using sodium hydroxide (36 %) as a base for the reaction was slightly higher than that obtained by using sodium carbonate (22 %) or potassium hydroxide (24 %). From these results, we optimized the conditions for the radiosynthesis of [18F]FMISO using [18F]5 as follows: 2 mg of precursor 6, 18 µmol of sodium hydroxide as a base for the reaction, and a reaction temperature of 150 °C for 20 min. After completion of the reaction, preparative HPLC for the reaction mixture was performed to efficiently separate [18F]FMISO from the mixture, affording the radiochemically and chemically pure product as an injectable solution [Fig. 5(A)]. No significant UV peak corresponding to unreacted 6 and its decomposition components were observed in the analytical HPLC chromatogram of the final product solution [Fig. 5(B)].

Fig. 4
figure 4

Radiochemical yields of [18F]FMISO via [18F]5 in varied precursor dose (A) and reaction temperature (B). [18F]5 was distilled from the reaction vial and was transferred to the next reaction vial containing 6 (0.5, 1.0, 2.0 and 4.0 mg) and 1 mol/L NaOH (4.5, 9.0, 18 and 36 µL) in anhydrous DMF (0.25 mL) maintained at -15 °C. After 2 min of trapping of [18F]5, the reaction mixture was heated at 90, 110, 130, and 150 °C for 20 min to obtain [18F]FMISO

Fig. 5
figure 5

Preparative HPLC (A) and analytical HPLC (B) chromatograms of [18F]FMISO by 18F-fluoroalkylation using [18F]5. The preparative HPLC conditions were as follows: XBridge C18 column (5 μm, 10 mm i.d. × 250 mm length; Waters), a mixture of ethanol and water (2:98, vol./vol.) as the mobile phase, 5.0 mL/min flow rate, and UV detection at 325 nm. The retention time of [18F]FMISO was approximately 12 min (A). The analytical HPLC conditions were as follows: XBridge C18 column (5 μm, 4.6 mm i.d. × 150 mm length; Waters), a mixture of 90 % acetonitrile solution and 50 mM ammonium phosphate buffer (pH 9.3) (7:93, vol./vol.) as the mobile phase, 1.0 mL/min flow rate, and UV detection at 325 nm. The retention time of [18F]FMISO was 5.6 min (B)

Table 1 summarizes the results of the automated radiosynthesis of [18F]FMISO by 18F-fluoroalkylation using 6 and [18F]5 for clinical applications. We successfully synthesized [18F]FMISO using [18F]5, with sufficient radioactivity (0.83 ± 0.2 GBq, n = 8) for clinical applications. The radiochemical yield of [18F]FMISO based on the cyclotron-produced [18F]F at the end of the synthesis (EOS) was 26 ± 7.5 % (n = 8). All the results of quality control for the [18F]FMISO injection complied with our in-house quality control and quality assurance specifications (Table 1).

Table 1 Radiosynthesis results of [18F]FMISO and [18F]PM-PBB3 by 18F-fluoroalkylation using precursor 6 and [18F]5

Automated radiosynthesis of [18F]PM-PBB3 using [18F]5

We synthesized [18F]PM-PBB3 by the 18F-fluoroalkylation of precursor 7 and [18F]5 (Fig. 3), according to the reaction conditions previously determined for the reaction of a conventional phenol precursor with [18F]5 (Fujinaga et al. 2018). After the trapping of [18F]5 for 2 min, the 18F-fluoroalkylation of 7 and [18F]5 was performed at 130 °C for 20 min. The reaction mixture was then separated using preparative HPLC [Fig. 6(A)] to produce radiochemically and chemically pure [18F]PM-PBB3 as an injectable solution [Fig. 6(B)].

Fig. 6
figure 6

Preparative HPLC (A) and analytical HPLC (B) chromatograms of [18F]PM-PBB3 by 18F-fluoroalkylation using [18F]5. The preparative HPLC conditions were as follows: Capcell Pak C18 column (5 μm, 10 mm i.d. × 250 mm length; Shiseido), a mixture of acetonitrile, water and triethylamine (40:60:0.1, vol./vol./vol.) as the mobile phase, 5.0 mL/min flow rate, and UV detection at 365 nm. The retention time of [18F]PM-PBB3 was 14.9 min (A). The analytical HPLC conditions were as follows: Atlantis T3 column (5 μm, 4.6 mm i.d. × 150 mm length; Waters), a mixture of acetonitrile and 50 mM ammonium acetate (40:60, vol./vol.) as the mobile phase, 1.0 mL/min flow rate, and UV detection at 365 nm. The retention time of [18F]PM-PBB3 was 12 min (B)

Table 1 summarizes the automated radiosynthesis results of [18F]PM-PBB3 by 18F-fluoroalkylation using precursor 7 and [18F]5 for clinical applications. We successfully synthesized [18F]PM-PBB3 using [18F]5, with sufficient radioactivity (0.79 ± 0.1 GBq, n = 11) for clinical applications. In addition, the radiochemical yield of [18F]PM-PBB3 based on the cyclotron-produced [18F]F at EOS was 16 ± 3.2 % (n = 11). All the results of quality control for the [18F]PM-PBB3 injection complied with our in-house quality control and quality assurance specifications (Table 1).

Discussion

We successfully synthesized [18F]FMISO and [18F]PM-PBB3 by 18F-fluoroalkylation using [18F]5 with sufficient radioactivity for clinical applications. For [18F]FMISO, the radiochemical yield of the 18F-fluoroalkylation of 6 with [18F]5 was 26 ± 7.5 % (n = 8, Table 1), while the yield by the direct 18F-fluorination of 1 with [18F]F was approximately 40 % (from the average result in our routine radiosynthesis). For [18F]PM-PBB3, the radiochemical yield of the 18F-fluoroalkylation of 7 with [18F]5 was 16 ± 3.2 % (n = 11, Table 1), whereas the yield obtained by the direct 18F-fluorination of 2 with [18F]F was 25 ± 6.0 % (n = 53) (Kawamura et al. 2021). The reason for the difference in radiochemical yields between the two methods is the relatively lower reactivity of the corresponding precursors toward [18F]5 as a radiolabeling agent than toward [18F]F. Moreover, the reactivity of [18F]5 seemed to be lower than that of conventional 18F-fluoroalkyl agents, such as [18F]fluoroethyl bromide and [18F]fluoroethyl iodide, toward the same phenol precursor. Recently, we found that the use of some Lewis acids could increase the reactivity of [18F]5 with aniline analogs (Fujinaga et al. 2019) and expect that the radiochemical yield of PET tracers containing the 18F-FHP moiety could be increased by 18F-fluoroalkylation using phenol or amine and [18F]5, catalyzed by a Lewis acid.

On the other hand, for direct 18F-fluorination, the tosylate precursors 1 and 2 should be synthesized with at least two steps from 6 to 7, respectively, and were limited to only the radiosynthesis of [18F]FMISO and [18F]PM-PBB3. For 18F-fluoroalkylation, imidazole precursor 6 and phenol precursor 7 are available and accessible. In particular, precursor 7 (PBB3) is an authentic unlabeled compound of [11 C]PBB3, which is a clinically used radiotracer for PET imaging of tauopathy in the human brain (Hashimoto et al. 2014, 2015; Maruyama et al. 2013). Moreover, 6 or 7 could be used to react with [18F]5 as well as other radiolabeling agents, such as [11 C]methyl iodide and 18F-fluoroalkyl agents, to produce diverse PET tracer candidates. A structure-activity relationship study is helpful for finding PET tracers with improved in vitro properties and in vivo behaviors by reacting the same precursor with diverse radiolabeling agents. This strategy has been applied to develop PET tracers in our group and to explore the best version from a series of candidates with the same chemical skeleton (Fujinaga et al. 2012; Zhang et al. 2003, 2004).

In this synthesis, the resulting [18F]5 radiolabeling agent was purified by distillation from an 18F-fluorinated mixture of epoxytosylate 8 with [18F]F, and was used for 18F-fluoroalkylation (Fujinaga et al. 2018). The distillation procedure removed all non-volatile impurities, such as metal ions from the cyclotron target, unreacted 8 and [18F]F, and the phase transfer reagent Kryptofix 222 and K2CO3. Because of the utilization of purified [18F]5, only a small amount of precursor 6 (2 mg) or 7 (1 mg) was used for the 18F-fluoroalkylation, resulting in a clear 18F-fluoroalkylated reaction mixture. As shown in the respective HPLC separation charts for the reaction mixtures, in addition to the unreacted [18F]5, only the desired product corresponding to [18F]FMISO [Fig. 5(A)] or [18F]PM-PBB3 [Fig. 6(A)] peak was obtained from the reaction. Because of the large difference in the retention times of [18F]5 and [18F]FMISO or [18F]PM-PBB3, HPLC separation was easily conducted to obtain two radiochemically and chemically pure products [Fig. 5(B) and 6(B)]. Moreover, after 18F-fluoroalkylation, the reaction mixture did not require deprotection with acid, directly resulting in [18F]FMISO or [18F]PM-PBB3.

For direct 18F-fluorination, the tosylate precursor 1 or 2 is not stable in the presence of excess K2CO3 and Kryptofix 222 at high temperatures; therefore, a relatively large amount of 1 (5 mg) (Tang et al. 2005) or 2 (2 mg) (Kawamura et al. 2021) was required for the 18F-fluorination with [18F]F in order to produce sufficient radioactivity for clinical applications. The unreacted precursors and decomposed chemical components made the HPLC purification inconvenient (Supplemental information: Fig. S2 and S3). In the synthesis of [18F]FMISO by the 18F-fluorination using 1 and [18F]F, after removal of the tosyl group in [18F]3 by treating the reaction mixture with HCl, p-toluenesulfonic acid (TsOH) was obtained. Only HPLC separation of the reaction mixture could not remove TsOH perfectly, and part of it would be left in the final product solution. Therefore, in our laboratory, after preparative HPLC for the reaction mixture of [18F]3 with HCl, the HPLC fraction was passed through a Sep-Pak cartridge (Cl form) to remove TsOH. In addition, [18F]FMISO was obtained as a chemically and radiochemically pure injectable solution.

Conclusions

In this study, we successfully synthesized [18F]FMISO and [18F]PM-PBB3 by the 18F-fluoroalkylation using [18F]5, although the radiochemical yields of the 18F-fluoroalkylation using [18F]5 were relatively lower than those of the corresponding direct 18F-fluorination using [18F]F. Although the radiochemical yields were slightly lower for the synthesis route, the 18F-fluoroalkylations with [18F]5 were cleaner and thus purification by HPLC alone yielded very pure products. Furthermore, we obtained relatively high chemical and radiochemical purity of [18F]FMISO or [18F]PM-PBB3 injection by radiosynthesis with the 18F-fluoroalkylation using [18F]5. Radiosynthesis using [18F]5 is expected to be widely used to develop and produce useful PET tracers containing the 18F-FHP moiety.

Availability of data and materials

Data are provided in the article and supplementary information.

Abbreviations

[18F]FMISO:

[18F]Fluoromisonidazole

[18F]PM-PBB3:

1-[18F]Fluoro-3-((2-((1E,3E)-4-(6-(methylamino)pyridine-3-yl)buta-1,3-dien-1-yl)benzo[d]thiazol-6-yl)oxy)propan-2-ol

[18F]5 :

[18F]Epifluorohydrin

HPLC:

High-performance liquid chromatography

PET:

Positron emission tomography

18F-FHP:

18F-3-Fluoro-2-hydroxy)propyl

PBB3:

2-((1E,3E)-4-(6-(Methylamino)pyridin-3-yl)buta-1,3-dienyl)benzo[d]thiazol-6-ol

K2CO3 :

Potassium carbonate

Kryptofix 222:

4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8,8,8]hexacosane

CH3CN:

Acetonitrile

NaOH:

Sodium hydroxide

DMF:

N,N-Dimethylformamide

TsOH:

p-Toluenesulfonic acid

EOS:

The end of synthesis

References

  • Arakawa R, Okumura M, Ito H, Seki C, Takahashi H, Takano H, et al. Quantitative analysis of norepinephrine transporter in the human brain using PET with (S,S)-18F-FMeNER-D2. J Nucl Med. 2008;49(8):1270–6.

    PubMed  Article  Google Scholar 

  • Bruehlmeier M, Roelcke U, Schubiger PA, Ametamey SM. Assessment of hypoxia and perfusion in human brain tumors using PET with 18F-fluoromisonidazole and 15O-H2O. J Nucl Med. 2004;45(11):1851–9.

    PubMed  Google Scholar 

  • Byun BH, Kim BI, Park SY, Ko IO, Lee KC, Kim KM, et al. Head-to-head comparison of 11 C-PiB and 18F-FC119S for Aβ imaging in healthy subjects, mild cognitive impairment patients, and Alzheimer’s disease patients. Med (Baltim). 2017;96(12):e6441.

    Article  Google Scholar 

  • Chung SJ, Yoon HJ, Youn H, Kim MJ, Lee YS, Jeong JM, et al. 18F-FEDAC as a targeting agent for activated macrophages in DBA/1 mice with collagen-induced arthritis: comparison with 18F-FDG. J Nucl Med. 2018;59(5):839–45.

    CAS  PubMed  Article  Google Scholar 

  • Cole EL, Stewart MN, Littich R, Hoareau R, Scott PJH. Radiosyntheses using fluorine-18: the art and science of late stage fluorination. Curr Top Med Chem. 2014;14(7):875–900.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Deng X, Rong J, Wang L, Vasdev N, Zhang L, Josephson L, et al. Chemistry for positron emission tomography: recent advances in 11 C-, 18F-, 13 N-, and 15O-labeling reactions. Angew Chem Int Ed Engl. 2019;58(9):2580–605.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Eschmann SM, Paulsen F, Reimold M, Dittmann H, Welz S, Reischl G, et al. Prognostic impact of hypoxia imaging with 18F-misonidazole PET in non-small cell lung cancer and head and neck cancer before radiotherapy. J Nucl Med. 2005;46(2):253–60.

    PubMed  Google Scholar 

  • Fujimura Y, Ikoma Y, Yasuno F, Suhara T, Ota M, Matsumoto R, et al. Quantitative analyses of 18F-FEDAA1106 binding to peripheral benzodiazepine receptors in living human brain. J Nucl Med. 2006;47(1):43–50.

    CAS  PubMed  Google Scholar 

  • Fujinaga M, Yamasaki T, Yui J, Hatori A, Xie L, Kawamura K, et al. Synthesis and evaluation of novel radioligands for positron emission tomography imaging of metabotropic glutamate receptor subtype 1 (mGluR1) in rodent brain. J Med Chem. 2012;55(5):2342–52.

    CAS  PubMed  Article  Google Scholar 

  • Fujinaga M, Ohkubo T, Yamasaki T, Zhang Y, Mori W, Hanyu M, et al. Automated synthesis of (rac)-, (R)-, and (S)-[18F]epifluorohydrin and their application for developing PET radiotracers containing a 3-[18F]fluoro-2-hydroxypropyl moiety. ChemMedChem. 2018;13(16):1723–31.

    CAS  PubMed  Article  Google Scholar 

  • Fujinaga M, Ohkubo T, Kumata K, Nengaki N, Zhang MR. Development of scandium-catalyzed N-[18F]fluoroalkylation of aryl and heteroaryl amines with [18F]epifluorohydrin. J Label Compd Radiopharm. 2019;62(Suppl 1):180.

    Google Scholar 

  • Fukumura T, Suzuki H, Mukai K, Zhang MR, Yoshida Y, Nemoto K, et al. Development of versatile synthesis equipment for multiple production of PET radiopharmaceuticals. J Label Compd Radiopharm. 2007;50(Suppl 1):202.

    Google Scholar 

  • Grierson JR, Link JM, Mathis CA, Rasey JS, Krohn KA. A radiosynthesis of fluorine-18 fluoromisonidazole. J Nucl Med. 1989;30(3):343–50.

    CAS  PubMed  Google Scholar 

  • Haneda E, Higuchi M, Maeda J, Inaji M, Okauchi T, Ando K, et al. In vivo mapping of substance P receptors in brains of laboratory animals by high-resolution imaging systems. Synapse. 2007;61(4):205–15.

    PubMed  Article  Google Scholar 

  • Harada R, Okamura N, Furumoto S, Furukawa K, Ishiki A, Tomita N, et al. 18F-THK5351: A novel PET radiotracer for imaging neurofibrillary pathology in Alzheimer disease. J Nucl Med. 2016;57(2):208–14.

    CAS  PubMed  Article  Google Scholar 

  • Harada R, Hayakawa Y, Ezura M, Lerdsirisuk P, Du Y, Ishikawa Y, et al. 18F-SMBT-1: a selective and reversible PET tracer for monoamine oxidase-B imaging. J Nucl Med. 2021;62(2):253–8.

    CAS  PubMed  Article  Google Scholar 

  • Hashimoto H, Kawamura K, Igarashi N, Takei M, Fujishiro T, Aihara Y, et al. Radiosynthesis, photoisomerization, biodistribution, and metabolite analysis of 11 C-PBB3 as a clinically useful PET probe for imaging of tau pathology. J Nucl Med. 2014;55(9):1532–8.

    CAS  PubMed  Article  Google Scholar 

  • Hashimoto H, Kawamura K, Takei M, Igarashi N, Fujishiro T, Shiomi S, et al. Identification of a major radiometabolite of [11 C]PBB3. Nucl Med Biol. 2015;42(12):905–10.

    CAS  PubMed  Article  Google Scholar 

  • Hsu JL, Lin KJ, Hsiao IT, Huang KL, Liu CH, Wu HC, et al. The imaging features and clinical associations of a novel tau PET tracer-18F-APN1607 in Alzheimer disease. Clin Nucl Med. 2020;45(10):747–56.

    PubMed  Article  Google Scholar 

  • Iwata R, Pascali C, Bogni A, Furumoto S, Terasaki K, Yanai K. [18F]Fluoromethyl triflate, a novel and reactive [18F]fluoromethylating agent: Preparation and application to the on-column preparation of [18F]fluorocholine. Appl Radiat Isot. 2002;57(3):347–52.

    CAS  PubMed  Article  Google Scholar 

  • Kämäräinen E-L, Kyllönen T, Nihtilä O, Björk H, Solin O. Preparation of fluorine-18-labelled fluoromisonidazole using two different synthesis methods. J Label Comp Radiopharm. 2004;47(1):37–45.

    Article  CAS  Google Scholar 

  • Kawamura K, Kumata K, Takei M, Furutsuka K, Hashimoto H, Ito T, et al. Efficient radiosynthesis and non-clinical safety tests of the TSPO radioprobe [18F]FEDAC: Prerequisites for clinical application. Nucl Med Biol. 2016;43(7):445–53.

    CAS  PubMed  Article  Google Scholar 

  • Kawamura K, Hashimoto H, Furutsuka K, Ohkubo T, Fujishiro T, Togashi T, et al. Radiosynthesis and quality control testing of the tau imaging positron emission tomography tracer [18F]PM-PBB3 for clinical applications. J Label Comp Radiopharm. 2021;64(3):109–19.

    CAS  Article  Google Scholar 

  • Lee BS, Chu SY, Kwon HR, Park C, Sirion U, Brockschnieder D, et al. Synthesis and evaluation of 6-(3-[18F]fluoro-2-hydroxypropyl)-substituted 2-pyridylbenzothiophenes and 2-pyridylbenzothiazoles as potential PET tracers for imaging Aβ plaques. Bioorg Med Chem. 2016;24(9):2043–52.

    CAS  PubMed  Article  Google Scholar 

  • Maruyama M, Shimada H, Suhara T, Shinotoh H, Ji B, Maeda J, et al. Imaging of tau pathology in a tauopathy mouse model and in Alzheimer patients compared to normal controls. Neuron. 2013;79(6):1094–108.

    CAS  PubMed  Article  Google Scholar 

  • McCarthy TJ, Dence CS, Welch MJ. Application of microwave heating to the synthesis of [18F]fluoromisonidazole. Appl Radiat Isot. 1993;44(8):1129–32.

    CAS  PubMed  Article  Google Scholar 

  • Miller PW, Long NJ, Vilar R, Gee AD. Synthesis of 11 C, 18F, 15O, and 13 N radiolabels for positron emission tomography. Angew Chem Int Ed Engl. 2008;47(47):8998–9033.

    CAS  PubMed  Article  Google Scholar 

  • Mori W, Takei M, Furutsuka K, Fujinaga M, Kumata K, Muto M, et al. Comparison between [18F]fluorination and [18F]fluoroethylation reactions for the synthesis of the PDE10A PET radiotracer [18F]MNI-659. Nucl Med Biol. 2017;55:12–8.

    CAS  PubMed  Article  Google Scholar 

  • Mori W, Yamasaki T, Fujinaga M, Ogawa M, Zhang Y, Hatori A, et al. Development of 2-(2-(3-(4-([18F]fluoromethoxy-d2)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione for positron emission tomography imaging of phosphodiesterase 10A in the brain. J Med Chem. 2019;62(2):688–98.

    CAS  PubMed  Article  Google Scholar 

  • Oh SJ, Chi DY, Mosdzianowski C, Kim JY, Gil HS, Kang SH, et al. Fully automated synthesis of [18F]fluoromisonidazole using a conventional [18F]FDG module. Nucl Med Biol. 2005;32(8):899–905.

    CAS  PubMed  Article  Google Scholar 

  • Sasaki T, Ito H, Kimura Y, Arakawa R, Takano H, Seki C, et al. Quantification of dopamine transporter in human brain using PET with 18F-FE-PE2I. J Nucl Med. 2012;53(7):1065–73.

    CAS  PubMed  Article  Google Scholar 

  • Su Y, Fu J, Yu J, Zhao Q, Guan, Zuo Y, et al. Tau PET imaging with [18F]PM-PBB3 in frontotemporal dementia with MAPT mutation. J Alzheimers Dis. 2020;76(1):149–57.

    PubMed  Article  Google Scholar 

  • Tagai K, Ono M, Kubota M, Kitamura S, Takahata K, Seki C, et al. High-contrast in vivo imaging of tau pathologies in Alzheimer’s and non-Alzheimer’s disease tauopathies. Neuron. 2021;109(1):42–58.

    CAS  PubMed  Article  Google Scholar 

  • Tago T, Furumoto S, Okamura N, Harada R, Adachi H, Ishikawa Y, et al. Structure-activity relationship of 2-arylquinolines as PET imaging tracers for tau pathology in Alzheimer disease. J Nucl Med. 2016;57(4):608–14.

    CAS  PubMed  Article  Google Scholar 

  • Tang G, Wang M, Tang X, Gan M, Luo L. Fully automated one-pot synthesis of [18F]fluoromisonidazole. Nucl Med Biol. 2005;32(5):553–8.

    CAS  PubMed  Article  Google Scholar 

  • Weng CC, Hsiao IT, Yang QF, Yao CH, Tai CY, et al. Characterization of 18F-PM-PBB3 (18F-APN-1607) uptake in the rTg4510 mouse model of tauopathy. Molecules. 2020;25(7):1750.

    CAS  PubMed Central  Article  Google Scholar 

  • Wilson AA, Dasilva JN, Houle S. Synthesis of two radiofluorinated cocaine analogues using distilled 2-[18F]fluoroethyl bromide. Appl Radiat Isot. 1995;46(8):765–70.

    CAS  PubMed  Article  Google Scholar 

  • Xie L, Yui J, Hatori A, Yamasaki T, Kumata K, Wakizaka H, et al. Translocator protein (18 kDa), a potential molecular imaging biomarker for non-invasively distinguishing non-alcoholic fatty liver disease. J Hepatol. 2012;57(5):1076–82.

    CAS  PubMed  Article  Google Scholar 

  • Yanamoto K, Kumata K, Yamasaki T, Odawara C, Kawamura K, Yui J, et al. [18F]FEAC and [18F]FEDAC: two novel positron emission tomography ligands for peripheral-type benzodiazepine receptor in the brain. Bioorg Med Chem Lett. 2009;19(9):1707–10.

    CAS  PubMed  Article  Google Scholar 

  • Yang Y, Wang X, Yang H, Fu H, Zhang J, Zhang X, et al. Synthesis and monkey-PET study of (R)- and (S)-18F-labeled 2-arylbenzoheterocyclic derivatives as amyloid probes with distinctive in vivo kinetics. Mol Pharm. 2016;13(11):3852–63.

    CAS  PubMed  Article  Google Scholar 

  • Yui J, Maeda J, Kumata K, Kawamura K, Yanamoto K, Hatori A, et al. 18F-FEAC and 18F-FEDAC: PET of the monkey brain and imaging of translocator protein (18 kDa) in the infarcted rat brain. J Nucl Med. 2010;51(8):1301–9.

    CAS  PubMed  Article  Google Scholar 

  • Zhang MR, Tsuchiyama A, Haradahira T, Yoshida Y, Furutsuka K, Suzuki K. Development of an automated system for synthesizing 18F-labeled compounds using [18F]fluoroethyl bromide as a synthetic precursor. Appl Radiat Isot. 2002;57(3):335–42.

    CAS  PubMed  Article  Google Scholar 

  • Zhang MR, Maeda J, Furutsuka K, Yoshida Y, Ogawa M, Suhara T, et al. [18F]FMDAA1106 and [18F]FEDAA1106: two positron-emitter labeled ligands for peripheral benzodiazepine receptor (PBR). Bioorg Med Chem Lett. 2003;13(2):201–4.

    CAS  PubMed  Article  Google Scholar 

  • Zhang MR, Maeda J, Ogawa M, Noguchi J, Ito T, Yoshida Y, et al. Development of a new radioligand, N-(5-fluoro-2-phenoxyphenyl)-N-(2-[18F]fluoroethyl-5-methoxybenzyl)acetamide, for PET imaging of peripheral benzodiazepine receptor in primate brain. J Med Chem. 2004;47(9):2228–35.

    CAS  PubMed  Article  Google Scholar 

  • Zhang MR, Suzuki K. [18F]Fluoroalkyl agents: synthesis, reactivity and application for development of PET ligands in molecular imaging. Curr Top Med Chem. 2007;7(18):1817–28.

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgements

We thank the staff of the Cyclotron Operation Section and the Department of Advanced Nuclear Medicine Sciences of the Institute for Quantum Medical Science for their support with the operation of the cyclotron and the production of the radioisotopes.

We would like to thank Editage (www.editage.jp) for English language editing.

Funding

This study was partly supported by Grants-in-Aid for Scientific Research (Basic Research B: 19H03610; 20H03635) from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.

Author information

Authors and Affiliations

Authors

Contributions

TO designed the study, performed synthesis, and analyzed the data; YK, MO, and NN operated the radiosynthesizer; MF, WM, KK, and MH assisted with the synthesis; KF and HH analyzed the quality control data; KK summarized the study, and wrote the manuscript; MRZ designed the study and reviewed the manuscript; all authors read and approved the final manuscript.

Corresponding author

Correspondence to Kazunori Kawamura.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no potential conflicts of interest with respect to the authorship or publication of this article.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Figure S1.

The system diagram of automated multi-purpose synthesizer developed in-house (Fukumura et al. 2007). Figure S2. The preparative HPLC chromatogram of [18F]FMISO synthesized by 18F-fluorination using 1 and [18F]F-, followed by the removal of the protecting group in [18F]3. The HPLC conditions were as follows: XBridge C18 column (5 μm, 10 mm i.d. × 250 mm length; Waters), with a mixture of ethanol and water (2:98, vol./vol.) as the mobile phase, a flow rate of 5.0 mL/min, and UV detection at 325 nm. Figure S3. The preparative HPLC chromatograms of [18F]PM-PBB3 synthesized by 18F-fluorination using 2 and [18F]F-, followed by the removal of the protecting group in [18F]4. The HPLC conditions were as follows: Capcell Pak C18 column (5 μm, 10 mm i.d. × 250 mm length; Shiseido, Tokyo, Japan), the mixture of acetonitrile, water and triethylamine (40:60:0.1, v/v/v) as the mobile phase, 5.0 mL/min flow rate, and UV detection at 365 nm. 

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ohkubo, T., Kurihara, Y., Ogawa, M. et al. Automated radiosynthesis of two 18F-labeled tracers containing 3-fluoro-2-hydroxypropyl moiety, [18F]FMISO and [18F]PM-PBB3, via [18F]epifluorohydrin. EJNMMI radiopharm. chem. 6, 23 (2021). https://doi.org/10.1186/s41181-021-00138-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s41181-021-00138-9

Keywords

  • 18F
  • [18F]Epifluorohydrin
  • [18F]FMISO
  • [18F]PM-PBB3
  • Positron emission tomography (PET)