Automated Radiosynthesis of cis- and trans-4-[18F]Fluoro-l-proline Using [18F]Fluoride

The positron emission tomography imaging agents cis- and trans-4-[18F]fluoro-l-proline are used for the detection of numerous diseases such as pulmonary fibrosis and various carcinomas. These imaging agents are typically prepared by nucleophilic fluorination of 4-hydroxy-l-proline derivatives, with [18F]fluoride, followed by deprotection. Although effective radiofluorination reactions have been developed, the overall radiosynthesis process is suboptimal due to deprotection methods that are performed manually, require multiple steps, or involve harsh conditions. Here we describe the development of two synthetic routes that allow access to precursors, which undergo highly selective radiofluorination reactions and rapid deprotection, under mild acidic conditions. These methods were found to be compatible with automation, avoiding manual handling of radioactive intermediates.

■ INTRODUCTION α-Amino acids are the key building blocks of life, acting as structural components of peptides and proteins. 1 They also play an important role in biochemical and physiological processes, including energy metabolism, and in the formation of neurotransmitters and hormones. Due to the varied and important roles of α-amino acids in nature, their structural analogues have often been used to study biological processes and mechanisms. 2,3 Positron emission tomography (PET) in combination with 18 F-labeled α-amino acids ( Figure 1) has been used for the non-invasive generation of molecular, functional, and metabolic information for a wide range of diseases. 4 Although most applications have focused on imaging various forms of cancer, compounds such as 6-[ 18 F]fluoro-L-DOPA have been used to investigate neurodegenerative disorders, including Parkinson's disease. 5 The cis and trans isomers of 4-[ 18 F]fluoro-L-proline, [ 18 F]1 and [ 18 F]2, respectively, have also been used to image a number of disease conditions. Proline and 4-hydroxyproline are major structural components of collagen (15−30%), and therefore, [ 18 F]1 and [ 18 F]2 have been used to investigate abnormal collagen biosynthesis in diseases such as liver cirrhosis, lung fibrosis, and various carcinomas. 4,6,7 Due to the importance of cis-and trans-4-[ 18 F]fluoro-Lproline ([ 18 F]1 and [ 18 F]2, respectively), several methods for the radiosynthesis of these compounds have been developed. 4c The most common approach involves the reaction of 4sulfonyloxy-L-proline derivatives with [ 18 F]fluoride, leading to fluorination with inversion of configuration (Scheme 1a). Development of the fluorination step by automation has resulted in fast and efficient reactions, while formation of the undesired diastereomer (usually as a minor product) can be controlled by reaction temperature and removed by HPLC at the end of the process. 8 The limitations of these approaches occur during the deprotection stage, which due to harsh conditions is performed manually. For example, removal of the Boc-protecting group and hydrolysis of the ester were done as  . 7a−c Deprotection has also been done using a two-step strategy involving acid-mediated removal of the Boc group (0.1 M HCl, 120°C), followed by hydrolysis of the methyl ester using sodium hydroxide. 7b In addition to requiring an extra step, alkaline hydrolysis of proline esters is known to produce side products, resulting in a decrease in the radiochemical yield (RCY). 7a For one of our imaging programs, we required access to cisand trans-4-[ 18 F]fluoro-L-proline ([ 18 F]1 and [ 18 F]2, respectively) as well as the nonradioactive analogues as standards for radiochemistry studies. Due to the limitations of previous approaches, we sought to develop a fully automated synthesis involving both a nucleophilic radiofluorination reaction and a single-step deprotection process. We now report the nonradioactive synthesis of both cis-and trans-4-fluoro-L-proline (1 and 2, respectively) from readily available (2S,4R)-N-Boc-4hydroxy-L-proline, using a deoxyfluorination reaction with morpholinosulfur trifluoride as the key step. Also described is a fully automated synthesis of [ 18 F]1 and [ 18 F]2, which combines a highly effective nucleophilic radiofluorination with a single-step deprotection (Scheme 1b).

■ RESULTS AND DISCUSSION
Our primary aim during this project was the design and synthesis of proline derivatives that would undergo clean and efficient nucleophilic fluorination reactions and that could be deprotected in a single step, under mild conditions. Previous syntheses of 4-fluoroprolines have generally used an Nprotected derivative of 4-hydroxyproline methyl ester as the starting material. 9,10 However, issues were reported during the nucleophilic fluorination step involving intramolecular participation of the ester carbonyl, which led to the formation of a fluoroproline byproduct (17%) with retention of configuration. 11 In this project, it was proposed that the use of a more bulky proline derivative, such as N-Boc-L-proline tert-butyl ester 5, would minimize any intramolecular reactions during the fluorination step. Furthermore, the use of two acidsensitive protecting groups would allow rapid and mild deprotection during the preparation of the 18 F-labeled targets.
Our synthesis of cis-4-fluoro-L-proline (1) began with the esterification of commercially available (2S,4R)-N-Boc-4hydroxy-L-proline (4) with O-tert-butyl-N,N-diisopropylisourea (Scheme 2). 12 This gave tert-butyl ester 5 in 68% yield. A precursor for radiofluorination studies and the synthesis of cis-4-[ 18 F]fluoro-L-proline [ 18 F]1 was prepared by tosylation of 5 under standard conditions. An initial attempt to complete the synthesis of cis-4-fluoro-L-proline (1) investigated the nucleophilic fluorination of tosyl derivative 6 using TBAF. 10d However, this led to elimination of tosic acid and the isolation of dehydroproline derivatives. Instead, (2S,4R)-hydroxy-Lproline derivative 5 was treated with morpholinosulfur trifluoride, and this allowed the single-step synthesis of 7 in 63% yield. Analysis of the 1 H NMR spectrum of the crude reaction material showed the presence of only the cis diastereomer, confirming complete inversion of configuration. This result suggests that the sterically encumbered tert-butyl ester prevents any intramolecular participation of the carbonyl and the formation of the undesired fluorinated trans diastereomer. 13 Acid-mediated deprotection of 7 using 2 M hydrochloric acid at room temperature gave after recrystallization cis-4-fluoro-L-proline (1) in 64% yield.
To access trans-4-fluoro-L-proline (2) using the same approach required the preparation of (2S,4S)-N-Boc-4hydroxy-L-proline (9). As (2S,4R)-N-Boc-4-hydroxy-L-proline (4) is readily available and inexpensive, we investigated a strategy for inversion of configuration of the 4-hydroxyl group. Previous methods have activated the 4R-hydroxyl group of (2S,4R)-4-hydroxy-L-proline ester derivatives by mesylation or using a Mitsunobu reaction, followed by inversion with benzoic acid and then hydrolysis of the resulting ester. 10c−f Raines and co-workers described a three-step approach involving hydroxyl group mesylation, inversion by intramolecular lactonization with the α-carboxylic acid, and then lactone hydrolysis. 10d Inspired by this, we developed a two-step approach in which lactone 8 was initially prepared by an intramolecular Mitsunobu reaction of (2S,4R)-N-Boc-4hydroxy-L-proline (4) (Scheme 3). 14 Lactone 8 was then hydrolyzed at room temperature with lithium hydroxide to give (2S,4S)-N-Boc-4-hydroxy-L-proline (9) in 71% yield over the two steps. This approach was scalable, allowing the multigram synthesis of 9. With the (2S,4S)-diastereomer 9 in hand, the The Journal of Organic Chemistry pubs.acs.org/joc Article same series of steps (tert-butyl esterification and tosylation) was used to access precursor 11. Similarly, reaction of 10 with morpholinosulfur trifluoride gave 4-fluoroproline 12 as a single diastereomer, and deprotection under mild acidic conditions gave trans-4-fluoro-L-proline (2) in good overall yield. The radiosynthesis of [ 18 F]1 and [ 18 F]2 using a TRACERlab FX FN automated synthesizer and precursors 6 and 11 was next investigated. During these experiments, no-carrier-added [ 18 F]fluoride from the cyclotron was trapped on a carbonatepreconditioned quaternary methylammonium (QMA) cartridge, eluted into the reactor with a solution containing K222/ K 2 CO 3 , and then azeotropically dried. To compare the effectiveness of precursors 6 and 11 with previous methods, [ 18 15 In addition, multiple runs on the synthesizer using triflic acid caused damage to tubing and values, resulting in leaks and failed syntheses.
Similar conditions for radiofluorination and subsequent deprotection of (2S,4R)-proline tert-butyl ester derivative 6 were then investigated ( Table 1, entry 1). To ensure complete conversion of [ 18 F]fluoride, a longer radiofluorination reaction time of 15 min was used. In addition, triflic acid was replaced with 2 M hydrochloric acid during the deprotection stage.
Following a total reaction time of 74 min, this gave [ 18 F]1 in a decay-corrected RCY of 42%. A benefit of a slightly longer radiofluorination reaction time was that less precursor was required for complete conversion of [ 18 F]fluoride. With precursor 6, the amount for each run could be reduced from 16 to 5 mg. The study next investigated the use of milder conditions to remove the acid-labile protecting groups. Radiofluorination of 6, followed by deprotection with 2 M hydrochloric acid at 60°C, gave [ 18 F]1 in 19% RCY (entry 2). It was proposed that the lower RCY for this production was partly due to the use of a strong cation exchange (SCX) cartridge during the final formulation stage. Therefore, the two-step process was repeated using both a shorter reaction time (5 min) for the deprotection step and a mixed-mode cation exchange (MCX) cartridge during the formulation (entry 3). This gave a 42% RCY of [ 18 F]1 after a total reaction time of 71 min. Further optimization was achieved by avoiding an evaporation stage after initial radiofluorination (entry 4). This resulted in a shorter overall reaction time of 63 min and gave [ 18 F]1 in 36% RCY. The corresponding radio-HPLC chromatogram under these optimized conditions showed clean synthesis of [ 18 F]1 ( Figure 2). The reaction mixture was found to contain 98.8% [ 18 F]1, with <0.4% trans isomer [ 18 F]2. 15,16 The optimized conditions were then used for the automated production, isolation, and purification of [ 18 F]1 (Scheme 5). After a total synthesis time of 59 min, this gave [ 18 F]1 in 41 ± 3.6% RCY (n = 9) with a >99% radiochemical purity. The molar activity of [ 18 F]1 was found to be >0.641 GBq μmol −1 . 17 The optimized conditions were then used for the automated synthesis of [ 18 F]2 using precursor 11. In a similar manner, the two-step process was found to be highly selective for the preparation of [ 18 F]2, generating the trans isomer in 97.7% yield, with 2.2% of the cis isomer also observed. 15 Use of this method for the automated production and purification of [ 18 F] 2 gave the target after a total synthesis time of 57 min, in 34 ± 4.3% RCY (n = 11) with a >99% radiochemical purity. The molar activity of [ 18 F]2 was found to be >0.320 GBq μmol −1 . 17 The stability of formulated products [ 18 F]1 and [ 18 F]2 using radio-HPLC was analyzed at 2 and 11 h points from the end of synthesis. 15 For both isomers, there was no observed radiochemical byproduct after 11 h, which confirmed that these imaging agents are stable within this time frame to decomposition pathways, such as epimerization or radiolysis.   . The bulky precursors underwent clean and reproducible radiofluorination, and the use of two acid-sensitive protecting groups allowed deprotection under mild conditions. It should be noted that both steps are highly amenable to automation when using a synthesizer and, thus, avoid typically harsh conditions and manual handling of radioactive intermediates.

■ EXPERIMENTAL SECTION
All reagents and starting materials were obtained from commercial sources and used as received unless otherwise stated. Dry solvents were purified using a solvent purification system. Brine refers to a saturated solution of sodium chloride. All reactions were performed in oven-dried glassware under an atmosphere of argon unless otherwise stated. All mixtures for reactions performed at increased temperatures were heated using an oil bath. Flash column chromatography was carried out using silica gel (40−63 μm). Aluminum-backed plates precoated with silica gel 60 (UV 254 ) were used for thin layer chromatography and visualized under ultraviolet light and by staining with KMnO 4 , ninhydrin, or vanillin. 1 H NMR spectra were recorded on an NMR spectrometer at 400 or 500 MHz, and data are reported as follows: chemical shift in parts per million relative to tetramethylsilane or the solvent as the internal standard (CDCl 3 , δ 7.26), multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet or overlap of nonequivalent resonances, integration). 13 C{ 1 H} NMR spectra were recorded on an NMR spectrometer at 101 or 126 MHz, and data are reported as follows: chemical shift in parts per million relative to tetramethylsilane or the solvent as the internal standard (CDCl 3 , δ 77.0), multiplicity with respect to hydrogen (deduced from DEPT experiments, C, CH, CH 2 , or CH 3 ). IR spectra were recorded on a FTIR spectrometer; wavenumbers are indicated in inverse centimeters. Mass spectra were recorded using electron impact or electrospray ionization techniques. HRMS spectra were recorded using a dual-focusing magnetic analyzer mass spectrometer. Melting points are uncorrected. Optical rotations were determined as solutions irradiating with the sodium D line (λ = 589 nm) using a polarimeter.
Analytical HPLC Method. Analytical HPLC was carried out on a Thermo Dionex Ulimate system 3000 equipped with a Berthold FlowStar LB 513 radio flow detector and a DAD-3000 UV detector. An isocratic mobile phase of 60% acetonitrile in water was used with a Phenomenex Luna 5 μm NH 2 100 Å, 250 mm × 4.6 mm column at a rate of 1 mL min −1 . The nonradioactive standards were detected using a UV wavelength of 210 nm.
cis-4-[ 18 F]Fluoro-L-proline [ 18 F]1. Cyclotron target water containing [ 18 F]fluoride was transferred to and trapped on a Sep-Pak QMA Carbonate Plus Light cartridge. The activity was eluted into a reaction vessel using a solution of Kryptofix 222 (15 mg) and potassium carbonate (2.4 mg) in acetonitrile (0.80 mL) and water (0.40 mL). This solution was dried by being stirred at 100°C under vacuum and a stream of helium gas for 2 min. This process was repeated twice using acetonitrile (2 × 1 mL). The [ 18 F]fluoride was then completely dried by applying full vacuum for 1 min. Di-tert-butyl (2S,4R)-4-(tosyloxy)pyrrolidine-1,2-dicarboxylate (6) (5.0 mg) in acetonitrile (1.0 mL) was added to the reaction vessel, which was sealed, and the mixture heated to 110°C for 15 min while being stirred. The reaction mixture was then cooled to 60°C, and a 4 M aqueous solution of hydrochloric acid (1.0 mL) was added (resulting in a 2 M concentration of hydrochloric acid). The reaction mixture was stirred at this temperature for 5 min and then concentrated by applying vacuum under a stream of helium gas. The resultant residue was then cooled to 30°C and diluted with a 50% aqueous solution of acetonitrile (2.0 mL). The reaction mixture was then transferred into the HPLC injector loop for purification. Purification was performed by semipreparative HPLC with a SYKMN S1122 solvent delivery system using a Phenomenex Luna 5 μm NH 2 100 Å, 250 mm × 10 mm column and eluted using a 60% aqueous solution of acetonitrile at a flow rate of 4 mL min −1 . The product fraction was identified using a gamma detector at a retention time of approximately 9 min and collected into a flask containing an aqueous solution (20 mL) adjusted to pH 3 using phosphoric acid. The diluted fraction was then passed onto an Oasis MCX Plus Short cartridge, washed with water (10 mL), and eluted from the cartridge with a 0.1 M aqueous solution of sodium phosphate (6.0 mL). The formulation was then adjusted to pH 7 by the addition of a 1 M aqueous solution of hydrochloric acid (0.5 mL). cis-4-[ 18 F]Fluoro-L-proline [ 18 F]1 was isolated in 41 ± 3.6% radiochemical yield with a radiochemical purity of >99% (n = 9). The total synthesis time from delivery of [ 18 F]fluoride to extraction of the product was 59 ± 1.9 min.
HPLC chromatograms and 1 H and 13 C{ 1 H} NMR spectra of all compounds (PDF)