Special Issue Review Received 30 April 2012,

Revised 14 November 2012,

Accepted 15 November 2012

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3007

Radiochemistry devoted to the production of monoamine oxidase (MAO-A and MAO-B) ligands for brain imaging with positron emission tomography† Ken Kersemans,* Nick Van Laeken, and Filip De Vos Monoamine oxidase (MAO) belongs to a family of flavin-containing integral enzymes that are present in the outer mitochondrial membrane in neurons and glial cells in the central nervous system. These enzymes catalyze the oxidative deamination of various neurotransmitters, biogenic amines, and xenobiotics, thereby influencing their availability and physiological activity in brain and body. Over the past decades, many potential positron emission tomography tracers have been put forward to visualize MAO in the brain with varying success, and recent publications on the topic illustrate the continuing interest in the field. The present review gives an overview of the compounds that have been put forward as possible MAO tracers in the brain and focuses on the radiochemical procedures that have been developed to produce them up till now. Relevant radioligands are grouped by the main radiochemical strategies that have been employed to synthesize them, and some interesting details and findings that are crucial to the radiosyntheses are provided. Keywords: monoamine oxidases; radiochemistry; brain imaging; PET

Introduction In 1938, Zeller et al. introduced the name ‘monoamine oxidase’.1 Monoamine oxidase (MAO) belongs to a family of flavin-containing integral enzymes that are present in the outer mitochondrial membrane in neurons and glial cells in the central nervous system, although a small proportion is associated with the microsomal fraction.2,3 In addition, it is found in liver, placenta, intestine, pancreas, and thrombocytes.4 Acting not only on primary but also on some secondary and tertiary amines, these enzymes catalyze the oxidative deamination of various neurotransmitters, biogenic amines, and xenobiotics, thereby influencing their availability and physiological activity in the brain and body.5–7 Oxidation is stoichiometrically accompanied by the reduction of oxygen to hydrogen peroxide (Eq. 1). MAO

RCH2 NHR’ þ H2 O þ O2 ! RCHO þ R’NH2 þ H2 O2

(1)

78

In mammals, two isoforms of the enzyme have been identified on the basis of their biochemical properties, substrate selectivity, and gene products. These isoforms, monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B), are encoded by separate genes that are closely linked on the X-chromosome and share 70% similarity in amino acid sequence.8 They are expressed in distinct cellular compartments within the central nervous system,7 and their relative appearance is species-specific.9 MAO-A occurs in catecholaminergic neurons as well as glia and preferentially oxidizes norepinephrine and serotonin.9,10 In human brain, MAO-B is predominant (70% of total brain activity) when compared with MAO-A, and it is localized in glial cells (highly abundant in astrocytes) and serotonergic neurons.9,11–13 MAO-B has

J. Label Compd. Radiopharm 2013, 56 78–88

mainly affinity for b-phenylethylamine and benzylamine.10,14 Both forms oxidize dopamine, tyramine, and octopamine.15 Also, different inhibitor specificities are noted for MAO-A and MAO-B. Monoamine oxidase B is a prospective target of molecular imaging biomarkers. On the basis of the localization of MAO-B in glial cells, mainly astrocytes, and the accumulation of the latter in neurodegenerative diseases and neuroinflammation, increased MAO-B binding of a positron emission tomography (PET) tracer can be indicative for the presence of inflammation in the brain.16–18 By this, tracing MAO-B can form a positive complement for tracers such as 2-deoxy-2-[18F]fluoro-D-glucose whose uptake is normally decreased in degenerative processes.9 This finding can be useful in the diagnosis of Alzheimer’s disease. In the Alzheimer brain, microglia and astrocytes are activated during neuroinflammation, and they accumulate in and around b-amyloid plaques.12 The observation that the MAO-B binding is higher in early Braak stages than in later Braak stages underlines the potency of the ligand as an early imaging biomarker. Furthermore, inhibition of MAO-B may reduce oxidative stress in the brain, by preventing

Laboratory for Radiopharmacy, Gent University, Gent, Belgium *Correspondence to: Ken Kersemans, Ghent University, Laboratory for Radiopharmacy, Harelbekestraat 72, B-9000 Gent, Belgium. E-mail: [email protected] †

This article is published in the Journal of Labeled Compounds and Radiopharmaceuticals as a special issue on Carbon-11 and fluorine-18 chemistry devoted to molecular probes for imaging the brain with PET, edited by Frédéric DOLLÉ, Service Hospitalier Frédéric Joliot Institut d’Imagerie BioMédicale, CEA 4 Place du Général Leclerc, F-91406 Orsay, France.

Copyright © 2013 John Wiley & Sons, Ltd.

K. Kersemans et al. Biography

Biography

Ken Kersemans was born in Turnhout, Belgium, in 1980. In 2003, he obtained his Master’s Degree in Analytical Chemistry, and in 2010, he graduated from the Free University of Brussels with a PhD in Chemistry. His doctoral research focused on the development of novel radiofluorinated alkylphenylalanine analogs for tumor imaging with positron emission tomography (PET). During his time as a doctoral student, he gained experience in the field of organic chemistry, radiochemistry, and quantum chemistry and gained insight in the preclinical evaluation of novel PET tracers and molecular imaging. He is currently a postdoctoral researcher at the University of Ghent, Belgium, where he continues to work on amino acid-based tumor tracers. Other research projects in which he is involved focus on the radiosynthesis of novel radiolabeled probes, preclinical drug development and the quantification of the immobilization of various radiolabeled biopolymers on functionalized biomaterials. His main interests are within the field of radiochemistry, radiopharmacy, and molecular imaging.

Filip De Vos was born in Ghent, Belgium in 1965. He obtained his PhD in Pharmaceutical Sciences at the University of Ghent where he also became head of the department of Radiopharmacy in the year 2005. Under his guidance, the department was further expanded and modernized with the establishment of the Infinity Lab (INnovative Flemish IN-vivo Imaging TechnologY), of which he is cofounder. His main interests are the development of new tracers for oncology and brain research, but because of the interdisciplinary nature of the department, he has gathered extensive experience in many related disciplines including pharmacy, radiopharmacy, radiochemistry, and medical imaging techniques.

Biography Nick Van Laeken was born in Bruges, Belgium, in 1988. In the autumn of 2006, he started his studies in pharmacy at the University of Ghent. After receiving his bachelor’s degree, he did his master thesis at the Ghent laboratory of Radiopharmacy concerning the in vitro and in vivo evaluation of humane 131 I-labeled antibodies for the detection of MT1-MMP, an integral membrane-bound proteinase that is overexpressed in several aggressive and metastatic tumors. In 2011, he graduated from the university with a Master’s Degree in Pharmaceutical Care. Three months later, he returned to the laboratory of Radiopharmacy to start his PhD. His research is focused within the field of neuroscience where he optimizes radiosynthetic procedures and uses positron emission tomography to contribute to the Ghent MRP project concerning the integrative neuroscience of behavioral control. Current interests include radiochemistry, radiopharmacy, medical imaging, kinetic modeling, and development of animal models relevant to the field of neurosciences.

J. Label Compd. Radiopharm 2013, 56 78–88

Radiosynthetic procedures Despite the multitude of radiopharmaceuticals dedicated to MAO imaging in the brain, the different radiochemical approaches to acquire them are rather limited and can be brought back to four main strategies. Two frequently used strategies for the introduction of the 11C-label employ either [11C]phosgene or [11C]methyl iodide (and more recently, [11C]methyl triflate). For the introduction of the 18F-label, usually the typical SN2 approach is employed, using no-carrier added 18F-fluoride, whereas rare reports exist on the use of electrophilic radiofluorination. The radiosynthesis of each compound will be discussed and grouped by synthon, and all relevant data on the type of precursor, total synthesis time, yield, specific activity, and corresponding reference are provided in summary in tables and figures. [11C]Phosgene as radiolabeling synthon The radiosynthetic procedures for the preparation of [11C]befloxatone, [11C]MD230254, and [11C]SL25.1188 make use of the radiochemical synthon [11C]phosgene as radioactive precursor to label the radioligand. [11C]Phosgene with high specific activity is usually prepared via [11C]CCl4 and iron-mediated conversion to [11C] COCl2. [11C]CCl4 itself arises from [11C]CH4 and elemental chlorine by radical reaction. However, the relative complexity of the associated radiosynthesis procedure and the use of the aggressive

Copyright © 2013 John Wiley & Sons, Ltd.

www.jlcr.org

79

the formation of hydrogen peroxide, which can be expected to slow the progression of Alzheimer’s disease.19 On the other hand, depression in nonsmokers has been linked to altered brain MAO-A activity levels, illustrating the clinical relevance of monitoring brain MAO-A activity levels.20 Fowler et al. in 2002 put forward three different approaches that have been used to selectively image MAO subtypes. First of all, there are the suicide inactivator radiotracers, for instance, the selective inhibitors [11C]clorgyline and [11C]L-deprenyl (MAO-A and MAO-B selectivity, respectively) or the nonselective inhibitor [11C]pargyline.10,21,22 After oxidation by MAO, these compounds

form radiolabeled reactive intermediates that are irreversibly, covalenty bound to the flavin cofactor of MAO. Another approach is found in metabolically trapped radiotracers. These types of radiotracers form, after oxidation by MAO, a charged labeled product that is intracellularly trapped. This is exemplified by the MAO-B substrate [11C]N,N-dimethylphenethylamine (DMPEA). After oxidation, [11C]CH3–NH+2 –CH3 is trapped in MAO-B-rich regions in the brain.23 Finally, reversible, subtype selective radiotracers such as [11C]harmine or [11C]SL25.1188 (MAO-A and MAO-B selectivity, respectively) make up the third approach towards the selective imaging of MAO subtypes.24,25 Over the past decades, many potential PET tracers have been put forward to visualize MAO in the brain with varying success, and recent publications on the topic illustrate the continuing interest in the field. An overview of the radiochemical procedures associated with those compounds is given in what follows.

K. Kersemans et al. chlorine gas limit its use. A comprehensive review on this topic has been published by Roeda and Dollé in 2010.26 The complexity and the need of automated radiosynthesis setups to produce formulations, suited for clinical applications of the described radioligands, represent the most important challenges to be overcome. [11C]Befloxatone An efficient radiosynthesis of [11C]befloxatone was reported by F. Dollé et al. (Figure 1) in 2003 that allows the preparation of large amounts of [11C]befloxatone for the purpose of multi injection PET protocols.27 The ring-opened precursor 2((R)-l-methoxy-3[[4-[(3R)-4,4,4-trifluoro-3-hydroxybutoxy]-phenyl]amino]-2-propanol) was labeled with no-carrier added [11C]phosgene. [11C]COCl2 was trapped (bubbling through the solution) at room temperature in CH2Cl2 containing the labeling precursor. The cyclization reaction of [11C]phosgene was fast and almost quantitative: [11C]befloxatone was formed in 90–95% radiochemical yield with a radiochemical and chemical purity of more than 99% within 20 min of radiosynthesis, including HPLC purification. The total decay-corrected radiochemical yield of [11C]befloxatone, on the basis of starting [11C]CH4, was 27–50%. The specific radioactivity measured at the end of the radiosynthesis was 18.5–74.0 GBq/mmol. Formulation of labeled product for intravenous injection was effected by evaporating the HPLC solvent, dissolution of the residue in physiological saline containing 10% of ethanol followed by sterile filtration. [11C]MD230254 The radiosynthesis of [11C]MD230254, using a custom made module, was described by Bernard et al. in 199628 and is shown in Figure 2. [11C]Phosgene was directly trapped at 15  C in a solution of the

precursor hydrazide in toluene, containing the precursor. At the end of the synthesis, the toluene was evaporated, and the crude residue was dissolved in ethyl acetate/methylene chloride 1.5/98.5 that was injected onto a semi-preparative HPLC. [11C]MD230254 was found to be lipophilic and adhered strongly to the flask, rendering dissolution in the mobile phase for HPLC problematic. To overcome this, 1,2-propanediol and phosphate buffer, pH 2.3, were added to the dissolving HPLC solvent, and the flask was gently stirred in a warm bath. After HPLC purification, the collected fraction containing [11C]MD230254 was evaporated and the crude residue was dissolved in sterile physiological saline solution. The specific radioactivity values obtained were between 11.1 and 22.2 GBq/mmol. [11C]SL25.1188 Figure 3 shows the direct labeling of SL25.1188 with [11C] phosgene according to the method described by Bramoullé et al. in 2008.29 [11C]Phosgene was trapped at room temperature for 1–2 min in dichloromethane containing the ring-opened precursor ((R)-1- methoxy-3-[[4-[(3R)-4,4,4-trifluoro-3-hydroxybutoxy]phenyl] amino]-2-propanol). The reaction was reported to feature a low yield with [11C]SL25.1188 being the only product aside from hydrolysis product(s) arising from unreacted [11C]phosgene. Heating the reaction mixture at 90–100  C did not significantly improve the yield nor did a larger amount of the labeling precursor increase the yield. The use of toluene, a suited reaction solvent for several similar reactions, was also unsuccessful. The most reproducible yields were observed using dichloromethane as the solvent and conditions including the use of 1.5–2.5 mg of the precursor (4.3–7.2 mmol), a short heating of the mixture (2 min at 100  C) followed by its

Figure 1. Radiosynthesis of [11C]befloxatone.

Figure 2. Radiosynthesis of [11C]MD230254.

80

Figure 3. Radiosynthesis of [11C]SL251188.

www.jlcr.org

Copyright © 2013 John Wiley & Sons, Ltd.

J. Label Compd. Radiopharm 2013, 56 78–88

K. Kersemans et al. concentration to dryness before final HPLC purification. The collected peak was formulated by means of a Sep-PakW preconcentration step (using a custom built device), and the final solution was made up with physiological saline to an ethanol concentration below 10%. The radiotracer preparation was found to be >95% chemically and radiochemically pure and radiochemically stable for at least 60 min. About 300–500 MBq of [11C]SL25.1188 could be obtained within 30–32 min with specific radioactivities ranging from 50 to 70 GBq/mmol (3.5–7% decay-corrected radiochemical yield, based on starting [11C]methane).

procedure, described previously for [11C]harmine. 4,9-Dihydro-lmethyl-3H-pyrido[3,4-b]indol-7-ol hydrochloride and 1.5 equivalents of aqueous sodium hydroxide were dissolved in DMSO, and the solution was shaken until a green color appeared. [11C]Methyl iodide was trapped at room temperature, and the reaction mixture was heated at 80  C for 5 min. After heating, the reaction mixture was treated as for [11C]harmine. Semi-preparative HPLC purification was performed on the crude reaction mixture. Methylation was achieved by using the respective sodium phenolate in DMSO, without the need for protection of amine functions. The total synthesis times amounted to 45 min.

[11C]Methyl iodide and [11C]methyl triflate as radiolabeling synthons

[11C]Brofaromin

The most prominent 11C-labeling method realized and applied in commercially available and automated radiosynthesis units uses [11C]methyl iodide as a radiolabel (Figure 4). Amines as well as thiol and phenolic hydroxyl functions can be directly radiolabeled to obtain the desired radioligand. [11C]Triflate has been introduced as an even faster methylating agent and is generally obtained by the reaction of [11C]methyl iodide with silver triflate. This methylating agent shows both a higher reactivity upon N-based, S-based, and O-based nucleophiles. Compounds that employ this synthon are summarized in Table 1. [11C]Harmine [11C]Harmine could be obtained in high radiochemical yield and specific radioactivity in a standardized and fully automated procedure. [11C]methyl iodide was trapped at room temperature in dimethylsulfoxide (DMSO) containing the precursor 7-hydroxyl-methyl-9H-pyrido[3,4-b]indole and one equivalent of 5 M aqueous sodium hydroxide. For the methylation, the respective sodium phenolate in DMSO was used, without need for protection of amine functions. The reaction mixture was heated at 80  C for 5 min, diluted with HPLC mobile phase, and injected onto a semi-preparative HPLC column. The fraction containing [11C] harmine was collected and transferred to a rotary evaporator to remove the solvent. The residue was redissolved in sterile 0.1 M phosphate buffer and sterile-filtered into a sterile ampule. [11C]Methylharmine [11C]Methylharmine could be obtained in high radiochemical yield and specific radioactivity by applying the methylation procedure used for [11C]harmine. 7-Methoxy-l-methyl-9H-pyrido [3,4-b]indole.HCl and aqueous potassium hydroxide (1.5 equivalents) were dissolved in dimethylformamide. After trapping of [11C] methyl iodide, the reaction mixture was heated at 80  C for 5 min and then treated as for [11C]harmine. Methylation was achieved by using the free amine base in dimethylformamide (DMF) solution. Purification by semi-preparative HPLC afforded the labeled compound in a radiochemical purity exceeding 98%. [11C]Harmaline [11C]Harmaline could be obtained in high radiochemical yield and specific radioactivity by methylation using the fully automated

J. Label Compd. Radiopharm 2013, 56 78–88

[11C]Clorgyline [11C]Methyl iodide was trapped at room temperature in a mixture of dimethylformamide and DMSO (3:1) containing the precursor N-[3-(2,4-dichlorophenoxy)propyl]-N-(2-propynyl)amine. The reaction mixture was heated at 80  C for 3 min and then treated and purified as for [11C]harmine. Methylation was achieved by using the free amine base in DMF solution. Purification by semi-preparative HPLC afforded the labeled compound in a radiochemical purity exceeding 98%. N,N-[11C]dimethylphenethylamine A mixture of N-methylphenylethylamine, [11C]methyl iodide, and aqueous NaOH in acetone was heated for 5 min at 70  C. The [11C]DMPEA produced was purified by normal phase HPLC using chloroform/methanol/ammonia (1500:50:1) as the solvent. The fraction containing [11C]DMPEA was collected and evaporated. The residue was dissolved in 10 mL of saline and passed through a sterile Millipore filter (0.22 mm). (R)-()-[11C]Deprenyl (R)-()-Deprenyl was labeled by reacting the secondary amine precursor nor-(R)-()-deprenyl with [11C]methyl triflate. [11C] methyl triflate was trapped at room temperature in acetonitrile containing 1.0 mg of precursor, and the labeling was then allowed to proceed for 1 min at 150  C in the presence of benzyltrimethylammonium hydroxide. The decay corrected yield, on the basis of [11C]methyl triflate, was 15%. The use of aqueous NaOH did not improve the yield except when higher base over precursor ratios were used. When 5 equivalents of aqueous NaOH were present in the reaction mixture, a 25% yield (decay corrected and based on [11C]methyl triflate) could be observed using similar conditions. The use of acetone as the solvent and only 1.5–2.0 equivalents of benzyltrimethylammonium hydroxide as the base gave similar results (25% decay-corrected yield, on the basis of [11C]methyl triflate). The use of DMF as the solvent, or when NaOH was used as the base, led to a decrease in the radiochemical yield. However, the best results were obtained with [11C]methyl triflate as the alkylating agent and 1.0 mg (4.8 mmol) of the precursor at 150  C for about 3 min in acetone

Copyright © 2013 John Wiley & Sons, Ltd.

www.jlcr.org

81

Figure 4. Schematic representation of isotopic methylation by means of methyl iodide and methyl triflate where X represents either an N, S, or O atom.

[11C]Brofaromin was prepared by trapping [11C]methyl iodide at room temperature in DMSO containing 7-bromo-2-(4-piperidinyl)5-hydroxybenzofuran and 1.5 equivalents of aqueous sodium hydroxide. The reaction mixture was heated at 110  C for 5 min and then treated and purified as for [11C]harmine. Methylation was achieved by using the respective sodium phenolate in DMSO, without the need for protection of amine functions. Purification by semi-preparative HPLC afforded the labeled compound in a radiochemical purity exceeding 98%.

K. Kersemans et al. Table 1. Synthesis of [11C]methyl substituted radioligands with [11C]methyl iodide or [11C]methyl triflate Synthesis time (min)

Radiochemical yield (%) decay corrected to EOB

[11C]Harmine30

43

72.5  3.6

18.0–87.3

[11C]Methylharmine30

45

83.8  7.4

18.0–87.3

[11C]Harmaline30

40

65.9  9.7

18.0–87.3

[11C]Brofaromin30

43

48.1  6.5

18.0–87.3

[11C]Clorgyline30

43

88.8  5.9

18.0–87.3

[11C]N,N-dimethyl phenethylamine31

35

20

>3.7

(R)-()-[11C]Deprenyl32

25

60

29.6–44.4

Radioligand, reference

ROH or RR0 NH precursor

Specific activity at EOS (GBq/mmol)

(Continues)

82 www.jlcr.org

Copyright © 2013 John Wiley & Sons, Ltd.

J. Label Compd. Radiopharm 2013, 56 78–88

K. Kersemans et al. Table 1. (Continued) Synthesis time (min)

Radiochemical yield (%) decay corrected to EOB

50–60

20–45

0.37–1.48

3-(4-[11C]-Methoxyphenyl)-6methyl-2H-1-benzopyran-2one3

40

18  7

110

N-((1-H-Pyrrol-2-yl)methyl)-N[11C]methylbenzylamine3

40

11  4

90

[11C]RS231534

35

28  4

25–92

[11C]RS236034

30

30  6

41–106

R or S [11C]ROMAO35

42

60–70

50

Radioligand, reference

ROH or RR0 NH precursor

[11C]Pargyline33

Specific activity at EOS (GBq/mmol)

EOB, End Of Bombardment; EOS, End Of Syntheses.

J. Label Compd. Radiopharm 2013, 56 78–88

observed yield was systematically lower (35% and 25%, respectively). The use of a large increase of base equivalents (3–5 equivalents) also gave lower yields (40% and 35%, respectively). When higher amounts of precursor were used (up to 5.0 mg), the final yield was not really affected, but the HPLC separation of the radiotracer (R)-()-[11C]deprenyl and the precursor was not successful.

Copyright © 2013 John Wiley & Sons, Ltd.

www.jlcr.org

83

containing 1.9 equivalents of aqueous NaOH. After labeling, the reaction mixture was concentrated to dryness and taken up in mobile phase and injected on HPLC. (R)-()-[11C]deprenyl could be synthesized in 25 min after End Of Bombardment (EOB), corresponding to a 60% decay corrected yield (on the basis of [11C]methyl triflate). Note that when less equivalents of base were used (1.3 or 1.6 base over precursor ratio), the

K. Kersemans et al. [11C]Pargyline [11C]CH3I was trapped in 1.5 mL acetone containing the precursor desmethylpargyline at dry-ice acetone temperature. The solution was then heated at 50–60  C for 5–10 min. Below a reaction temperature of 60  C, most of the methylated product was [11C] pargyline. At 65  C, several radioactive impurities were observed. Unreacted [11C]CH3I was swept with a helium stream at 200 mL/min for 2–3 min at 50–60  C. Acetone was also removed with a helium stream. The residue was dissolved in 60/40 MeOH/H2O and subjected to HPLC. The [11C]pargyline fraction was collected, acidified with a few drops of HCl, and evaporated to dryness. [11C]Pargyline was obtained with a radiochemical purity of over 99%. The [11C]pargyline was dissolved in saline, neutralized with 0.1 M NaOH, and filtered through a membrane filter (0.22 mm). As is pointed out by Ishiwata et al.,33 the acidification and subsequent evaporation of the solvents are the most critical steps, and part of the [11C]pargyline degrades if too much HCl is added. Despite this observation, the authors do not provide any further details on how to avoid this degradation. 3-(4-[11C]-Methoxyphenyl)-6-methyl-2H-1-benzopyran-2-one Preparation of 3-(4-[11C]-methoxyphenyl)-6-methyl-2H-1-benzopyran2-one was accomplished via the ‘LOOP’ method,36 according to a modified literature procedure.37 The precursor, 3-(4-hydroxyphenyl)6-methyl-2H-1-benzopyran-2-one, was dissolved in DMF and tetrabutylammonium hydroxide and injected into a clean, dry loop. The [11C]CH3I was then introduced, and the mixture was reacted for 5 min. The reaction mixture was then purified via semipreparative HPLC. The fraction containing the major radiochemical product (tR = 13.8 min) was collected and loaded on a t-C18 plus Sep-PakW cartridge. Finally, 3-(4-[11C]-methoxyphenyl)-6-methyl2H-1-benzopyran-2-one was eluted from the t-C18 plus Sep-PakW cartridge with 1 mL EtOH, followed by the addition of 10 mL sterile 0.9% saline.38 The HPLC analysis of formulated 3-(4-[11C]methoxyphenyl)-6-methyl-2H-1-benzopyran-2-one revealed a high radiochemical (>98%) purity. N-((1-H-Pyrrol-2-yl)methyl)-N-[11C]methylbenzylamine N-((1-H-Pyrrol-2-yl)methyl)-N-[11C]methylbenzylamine was synthesized using the same procedure as described in the section about 3-(4-[11C]-Methoxyphenyl)-6-methyl-2H-1-benzopyran-2-one. N-((1-H-pyrrol-2-yl)methyl)-N-[11C]methylbenzylamine was dissolved in DMF and injected into a clean, dry loop. The radiolabeled [11C] CH3I was then introduced, and the mixture was reacted for 5 min. The reaction mixture was then purified via semi-preparative HPLC. The major radiochemical peak was collected and formulated as described for 3-(4-[11C]-methoxyphenyl)-6-methyl-2H-1benzopyran-2-one. The HPLC analysis of formulated N-((1-Hpyrrol-2-yl)methyl)-N-[11C]methylbenzylamine revealed a high radiochemical (>98%) purity.3 N-(Phenethyl)-N-[11C]methyl-1H-pyrrole-2-carboxamide ([11C]RS2315)

84

A solution of precursor N-(phenethyl)-1H-pyrrole-2-carboxamide (RS2115) in DMSO/DMF and aqueous tetrabutylammonium hydroxide was added to a reaction vessel, and the mixture was cooled in an ice bath. A stream of helium containing the alkylating agent [11C]CH3I was bubbled through the reaction mixture, and RS2115 was chemoselectively methylated. The reaction mixture was heated in an oil bath at 65  C for 10 min, subsequently diluted with HPLC eluent and purified with semi-preparative C18 HPLC. The fraction containing [11C]RS2315 was collected and preconcentrated on a C18 Sep-Pak. After the cartridge had been washed with

www.jlcr.org

sterile water, the desired product, [11C]RS2315, was eluted with ethanol and diluted with saline. (R)-N-(a-Cyclohexylethyl)-N-[11C]methyl-1H-pyrrole-2-carboxamide ([11C]RS2360) The same procedure as described for [11C]RS2315, with minor modifications, was applied to synthesize [11C]RS2360. [11C]RS2360 was prepared by chemoselective methylation with [11C]CH3I of the normethylderivate (R)-N-(a-cyclohexylethyl)-1H-pyrrole-2carboxamide (RS2226) in DMF/DMSO for 7 min at 70  C using aqueous Bu4NOH as a base. Purification was performed by semipreparative HPLC. The collected HPLC fraction containing [11C] RS2360 was diluted with Dulbecco’s phosphate buffered saline (pH 7.4) and preconcentrated on a C18 Sep-Pak. After the cartridge was washed with 5 mL of sterile water, the desired product, [11C] RS2360, was eluted with EtOH and diluted with physiological saline. (R)-()- and (S)-(+)-1-(1-[11C]methyl-1H-pyrrol-2-yl)-2-phenyl-2(1-pyrrolidinyl)ethanone (R- and S-[11C]ROMAO) The cyclotron-produced [11C]CH3I was bubbled through a suspension containing the precursor, (R)-()- or (S)-(+)-2-phenyl1-(1H-pyrrol-2-yl)-2-(1-pyrrolidinyl)ethanone (desmethyl-R- and SROMAO) and NaH in DMF. The reaction mixture was left for 1 min at room temperature before it was quenched with HPLC eluent and transferred into the HPLC loop. The reaction mixture was purified using a semi-preparative HPLC. The fraction corresponding to the labeled product was collected, concentrated to ca. 5 mL under reduced pressure at 90  C, and formulated with isotonic saline solution. [18F]Fluoride as radiolabeling synthon Chemistry with 18F sources has been widely developed, and today this chemistry offers a wide range of methods and applications.39 Chemistry with nucleophilic [18F]fluoride (Figure 5) is frequently performed because of its ease of [18F]fluoride preparation and use and the typically high specific activities resulting from these radiochemical reactions. Methods of radionuclide incorporation range from aromatic and aliphatic substitution to the use of labeled prosthetics. Also, although less favored today, the use of electrophilic [18F]F2 has been successful in synthesizing a host of labeled compounds.40 MAO ligands that employ [18F]fluoride as radiolabeling synthon are summarized in Table 2. [18F]N-(2-aminoethyl)-5-fluoro-2-pyridinecarboxamide Both electrophilic and nucleophilic radiosynthesis strategies have been investigated for the production of [18F]N-(2-aminoethyl)-5fluoro-2-pyridinecarboxamide. The latter method is preferred as electrophilic radiofluorination leads to radiochemically impure [18F]N-(2-aminoethyl)-5-fluoro-2-pyridinecarboxamide. N-[2-(tbutylcarbamoyl)ethyl]-5-nitropyridine-2-carboxamide was labeled with [18F]fluoride, in the presence of KryptofixW2.2.2 (K2.2.2) and potassium carbonate in DMSO for 20 min at 135  C, according to the 3,4-dihydroxy-6-18F-fluoro-L-phenylalanine (FDOPA) method, reported by Reddy et al. (1993).41 The reaction mixture was then hydrolyzed using 20% HCI for 10 min at 135  C. The hydrolyzed final product was neutralized with NaOH and purified using an HPLC system. The product fraction was collected and

Figure 5. Schematic representation of radiofluorination by means of radiofluoride where X represents a suitable leaving group.

Copyright © 2013 John Wiley & Sons, Ltd.

J. Label Compd. Radiopharm 2013, 56 78–88

K. Kersemans et al. Table 2. Synthesis of [18F]fluoride substituted radioligands with [18F]fluoride Synthesis time (min)

Radiochemical yield (%) decay corrected to EOB

[18F]N-(2-aminoethyl)-5fluoro-2pyridinecarboxamide41

120

10

Unspecified

[18F]HMP (N-(6-[18F]fluorohexyl)-Nmethylpropargylamine)3

35

29  5

37

7-(2-[18F]fluoroethoxy)-1methyl-9H-b-carboline36

70

23  3

605  110

7-(3-[18F]fluoro-propoxy)-1methyl-9H-b-carboline36

70

10  2

744  30

7-[2-(2-[18F]fluoroethoxy) ethoxy]-1-methyl-9H-bcarboline36

70

12  3

508  160

7-{2-[2-(2-[18F]fluoroethoxy) ethoxy]-ethoxy}-1-methyl9H-b carboline36

70

14  4

440  100

Radioligand and reference

Precursor

Specific activity at EOS (GBq/mmol)

(Continues)

85

J. Label Compd. Radiopharm 2013, 56 78–88

Copyright © 2013 John Wiley & Sons, Ltd.

www.jlcr.org

K. Kersemans et al. Table 2. (Continued) Synthesis time (min)

Radiochemical yield (%) decay corrected to EOB

(2R)-N-propargyl-N-(2-[18F] fluoroethyl)-1phenylpropan-2-amine13

70–80

68

280

(1S,2R)-N-propargyl-1-[18F] fluoro-N-methyl-1phenylpropan-2-amine13

70–80

41

240

(2S)-N-methyl-N-propargyl1-[18F]fluoro-3phenylpropan-2-ylamine13

70–80

46

310

DL-4-[18F]Fluorodeprenyl42

90

11

21.1

75–80

40–70

>200

Radioligand and reference

Precursor

[18F]Fluororasagiline43

buffered with 0.6 M phosphate buffer resulting in an isotonic and injectable radiopharmaceutical. N-(6-[18F]Fluorohexyl)-N-methylpropargylamine ([18F]HMP)

86

The synthesis of [18F]HMP (N-(6-[18F]fluorohexyl)-N-methylpropargylamine) was effectively prepared by nucleophilic 18F-fluorination of the precursor, N-(6-bromohexyl)-N-methylpropargylamine40 by Mukherjee J et al. but seemed hard to reproduce. Therefore, an alternative precursor bearing a tosyloxy leaving group, namely 6-(methyl(prop-2-ynyl)amino)hexyl 4-methylbenzenesulfonate, was prepared by N. Vasdev et al. The radiosynthesis was carried out via general automated methods using a GE FXFN radiofluorination module as reported by van Oosten et al.,38 with only minor modifications. The precursor, 6-(methyl(prop-2-ynyl) amino)hexyl 4-methylbenzenesulfonate, dissolved in DMSO was added to the reaction vessel containing dried potassium [18F]fluoride-kryptofix 2.2.2 (K[18F]F-K222) complex. The reaction mixture was heated to 90  C for 5 min and quenched with H2O.

www.jlcr.org

Specific activity at EOS (GBq/mmol)

The reaction mixture was then purified via semi-preparative HPLC. The major radiochemical peak was collected and formulated as described by van Oosten et al.,38 The HPLC analysis of the formulated compound revealed high radiochemical (>99%) purities. 18

F-b-Carboline alkaloids

Several 18F-b-carboline alkaloids were labeled by means of an identical radiochemical procedure: the precursor was dissolved in dry DMF and added to a solution of the dried K[18F]F-K222 complex in dry DMF. The reaction mixture was heated at 150  C for 15 min in a heating block, then diluted with water and purified by semi-preparative HPLC. The isolated compound was dissolved in phosphate buffer (pH 7.4)/PEG 4:1. Novel fluorine-18-labeled analogs of L-deprenyl The precursors for N-[-(2S)-1-[18F]fluoro-3-phenylpropan-2-yl]-Nmethylprop-2-yn-1-amine, (2R)-N-propargyl-N-(2-[18F]fluoroethyl)-1-phenylpropan-2-amine, or (1S,2R)-N-propargyl-1-[18F]

Copyright © 2013 John Wiley & Sons, Ltd.

J. Label Compd. Radiopharm 2013, 56 78–88

K. Kersemans et al. fluoro-N-methyl-1-phenylpropan-2-amine were dissolved in DMSO and added to the vial containing dried K[18F]F-K222 complex. The closed reaction vessel was then heated at 120  C for 20 min. The reaction vessel was cooled to room temperature and diluted with 18 MΩ H2O before injecting to the HPLC. All three fluorine18-labeled radioligands were purified by reverse phase HPLC. The fraction of the desired compounds was collected and evaporated to dryness. The residue was dissolved in sterile phosphate buffered saline (PBS; pH = 7.4) and filtered through a sterile filter, yielding a sterile and pyrogen-free solution of the radioligand. The radiochemical purity was >99% for all three compounds. DL-4-[18F]Fluorodeprenyl No-carrier-added DL-a-methyl-b-4-[18F]fluorophenyl-N-methyl-Npropynylethylamine (DL-4-[18F]fluorodeprenyl) was synthesized via the following three-step procedure: (1) nucleophilic aromatic substitution by [18F]fluoride on 4-nitrobenzaldehyde to produce 4-[18F]fluorobenzaldehyde (yield 65%); (2) the reaction of 4-[18F] fluorobenzaldehyde with (1-chloro-1-(trimethylsilyl)ethyl)lithium followed by hydrolysis to give 4-[18F]fluorophenylacetone (yield 50%); and (3) reductive alkylation of 4-[18F]fluorophenylacetone with N-methyl-propynylamine in the presence of NaBH3CN (yield 35%) followed by HPLC purification to give a racemic mixture of 4-[18F]fluorodeprenyl. This synthesis approach, the conversion of an aromatic aldehyde to a homologous methyl ketone, extends the flexibility of the nucleophilic aromatic substitution reaction by applying it to the synthesis of radiotracers that do not bear electron-withdrawing activating groups on the aromatic ring. [18F]Fluororasagiline The precursor (3aS,8aR)-3-[prop-2-yn-1-yl]-3,3a,8,8a-tetrahydroindeno [1,2-d][1,2,3]oxathiazole 2,2-dioxide (0.02 mmol, 5 mg) was dissolved in DMSO (500 mL) and was added to the reaction vial containing dried K[18F]F-K222 complex. The closed reaction vessel was heated at 130  C for 20 min and cooled down to room temperature. Different acids such as sulfuric acid, trifluoroacetic acid, hydriodic acid, and hydrochloric acid were tested for the hydrolysis of the sulfamidate group. Trifluoroacetic acid and hydriodic acid did not result in the formation of any hydrolyzed product, whereas the use of sulfuric acid gave the hydrolyzed product with a mixture of undesired radiolabeled compounds. Hydrolysis with hydrochloric acid (200 mL of 0.5 N) gave the highest yield at 110  C for 20 min. The reaction mixture was cooled to room temperature and was generally diluted with water to a total volume of 2 mL before injecting to the HPLC for purification. A radioactive fraction corresponding the pure [18F]fluororasagiline was collected and diluted with 50 mL ultrapure water. The resulting mixture was loaded onto a preconditioned Sep-Pak tC18 plus cartridge. The cartridge was washed with water (10 mL), and the isolated product, [18F]fluororasagiline, was eluted with 1 mL of ethanol in a sterile vial containing phosphate buffer saline solution (PBS, 9 mL) to give the product in an isolated yield 40–70% corrected for decay, within 75–80 min and a purity of >99%.

Summary and outlook

J. Label Compd. Radiopharm 2013, 56 78–88

Conflict of Interest The authors have declared that there is no conflict of interest.

References [1] E. A. Zeller, Helv. Chim. Acta 1938, 21, 880–890. [2] M. B. Youdim, D. Edmondson, K. F. Tipton, Nat. Rev. Neurosci. 2006, 7, 295–309. [3] N. Vasdev, O. Sadovski, M. D. Moran, J. Parkes, J. H. Meyer, S. Houle, A. A. Wilson, Nucl. Med. Biol. 2011, 38, 933–943. [4] A. Buck, L. D. Frey, P. Bläuenstein, G. Krämer, A. Siegel, B. Weber, P. A. Schubiger, H. G. Wieser, Eur. J. Nucl. Med. 1998, 25, 464–470. [5] T. P. Singer and R. R. Ramsay, FASEB J. 1995, 9, 605–610. [6] J. G. Richards, J. Saura, J. Ulrich, M. Prada, Psychopharmacology 1992, 106, S21–S23. [7] J. C. Shih, K. Chen, M. J. Ridd, Annu. Rev. Neurosci. 1999, 22, 197–217. [8] A. W. J. Bach, N. C. Lan, D. J. Bruke, C. W. Abell, M. E. Bembenek, S. W. Kwan, P. H. Seeburg, J. C. Shih, FASEB J. 1988, 2, A1733–A1733. [9] J. S. Fowler, J. Logan, N. D. Volkow, G. J. Wang, R. R. MacGregor, Y. S. Ding, Methods 2002, 27, 263–277. [10] J. P. Johnston, Biochem. Pharmacol. 1968, 17, 1285–1297. [11] P. Riederer, C. Konradi, V. Schay, E. Kienzl, G. Birkmayer, W. Danielczyk, E. Sofic, M. B. Youdim, Adv. Neurol. 1987, 45, 111–118. [12] B. Gulyás, E. Pavlova, P. Kása, K. Gulya, L. Bakota, S. Várszegi, E. Keller, M. C. Horváth, S. Nag, I. Hermecz, K. Magyar, C. Halldin, Neurochem. Int. 2011, 58, 60–68. [13] S. Nag, L. Lehmann, T. Heinrich, A. Thiele, G. Kettschau, R. Nakao, B. Gulyás, C. Halldin, J. Med. Chem. 2011, 54, 7023–7029. [14] V. Pérez, J. L. Marco, E. Fernández-Alvarez, M. Unzeta, Brit. J. Pharmacol. 1999, 127, 869–876. [15] M. B. Youdim, P. Riederer, J. Neural Transm. Gen. Sect. 1993, 91, 181–95. [16] J. Saura, J. M. Luque, A. M. Cesura, M. Da Prada, V. Chan-Palay, G. Huber, J. Löffler, J. G. Richards, Neuroscience 1994, 62, 15–30. [17] J. Ekblom, S. S. Jossan, L. Oreland, E. Walum, S. M. Aquilonius, J. Neural Transm-Supp. 1994, 41, 253–258. [18] S. F. Carter, M. Schöll, O. Almkvist, A. Wall, H. Engler, B. Långström, A. Nordberg, J. Nucl. Med. 2012, 53, 37–46. [19] J. Hirvonen, M. Kailajärvi, T. Haltia, S. Koskimies, K. Någren, P. Virsu, V. Oikonen, H. Sipilä, P. Ruokoniemi, K. Virtanen, M. Scheinin, J. O. Rinne, Clin. Pharmacol. Ther. 2009, 85, 506–512. [20] J. H. Meyer, N. Ginovart, A. Boovariwala, S. Sagrati, D. Hussey, A. Garcia, T. Young, N. Praschak-Rieder, A. A. Wilson, S. Houle, Arch. Gen. Psychiatry 2006, 63, 1209–1216. [21] J. Knoll, K. Magyar, Adv. Biochem. Psychopharmacol. 1972, 5, 393–408. [22] K. Ishiwata, T. Ido, K. Yanai, K. Kawashima, Y. Miura, M. Monma, S. Watanuki, T. Takahashi, R. Iwata, J. Nucl. Med. 1985, 26, 630–636. [23] O. Inoue, T. Tominaga, T. Yamasaki, H. Kinemuchi, J. Neurochem. 1985, 44, 210–216. [24] M. Bottlaender, H. Valette, J. Delforge, W. Saba, I. Guenther, O. Curet, P. George, F. Dollé, M. C. Grégoire, J. Cerebr. Blood. F. Met. 2010, 30, 792–800.

Copyright © 2013 John Wiley & Sons, Ltd.

www.jlcr.org

87

Positron emission tomography has been applied as a useful tool for imaging brain MAO activity in humans, and a multitude of radioligands have been developed in order to study both MAO-A and MAO-B activity and density by PET. Among those radioligands, [11C]L-deprenyl is the one most widely used in clinical and preclinical studies. However, the short half life of carbon-11 (20.4 min)

makes carbon-11-labeled tracers not suitable for distribution to PET centers not equipped with an on-site cyclotron. Another major drawback of [11C]L-deprenyl is that its main radiometabolite [11C]L-methamphetamine enters the brain. As a consequence, there is still a high interest in the development of labeled MAO inhibitors with longer half-life as biological probes to map MAO activity in brain, and as recent publications indicate, continuing efforts are made to find a radioligand, which combines the benefits of the 18F-label with the optimal biological behavior and that can be produced by means of automated synthesis modules in sufficient amounts to allow clinical application. Of all currently available radioligands for imaging the brain, MAO activity in humans [11C]Clorgyline (MAO-A) and [11C]L-deprenyl (MAO-B) are still the most widely used ligands. However, the promising initial results that were obtained with the newly described [18F]fluororasagiline might open up new vistas for imaging MAO activity in the brain.

K. Kersemans et al. [25] W. Saba, H. Valette, M. A. Peyronneau, Y. Bramoullé, C. Coulon, O. Curet, P. George, F. Dollé, M. Bottlaender, Synapse 2010, 64, 61–69. [26] D. Roeda, F. Dollé, Curr. Top. Med. Chem. 2010, 10, 1680–700. [27] F. Dollé, Y. Bramoullé1, F. Hinnen, S. Demphel, P. George, M. Bottlaender, J. Label. Compd. Radiopharm. 2003, 46, 783–792. [28] S. Bernard, C. Fuseau, L. Schmid, R. Milcent, C. Crouzel, Eur. J. Nuc. Med. 1996, 23, 150–156. [29] Y. Bramoullé, F. Puech, W. Saba, H. Valette, M. Bottlaender, P. George, F. Dolle, J. Label. Compd. Radiopharm. 2008, 51, 153–158. [30] M. Bergström, G. Westerberg, T. Kihlberg, B. Langström, Nucl. Med. Biol. 1997, 24, 381–388. [31] H. Shinotoh, O. Inoue, K. Suzuki, T. Yamasaki, M. lyo, K. Hashimoto, T. Tominaga, T. Itoh, Y. Tateno, H. Ikehira, J. Nucl. Med. 1987, 28, 1006–1011. [32] F. Dolle, Y. Bramoulle, F. Hinnen, J. S. Fowler, J. Label. Compd. Radiopharm. 2002, 45, 803–811. [33] K. Ishiwata, T. Ido, K. Yanai, K. Kawashima, Y. Miura, M. Monma, S. Watanuki, T. Takahashi R. Iwata, J. NucI. Med. 1985, 26, 630–636.

[34] S. De Bruyne, G. La Regina, S. Staelens, L. Wyffels, S. Deleye, R. Silvestri, F. De Vos, Nucl. Med. Biol. 2010, 37, 459–467. [35] S. B. Jensen, R. Di Santo, A. K. Olsen, K. Pedersen, R. Costi, R. Cirilli, P. Cumming, J. Med. Chem. 2008, 51, 1617–1622. [36] E. Blom, F. Karimi, O. Eriksson, H. Hall, B. Langström, J. Label. Compd. Radiopharm. 2008, 51, 277–282. [37] J. S. Fowler, G. J. Wang, J. Logan, S. Xie, N. D. Volkow, R. R. MacGregor, D. J. Schlyer, N. Pappas, D. L. Alexoff, C. Patlak, A. P. Wolf, J. NucI. Med. 1995, 36, 1255–1262. [38] E. M. van Oosten, A. A. Wilson, K. A. Stephenson, D. C. Mam, B. G. Polloc, B. H. Mulsant, Appl. Radiat. Isot. 2009, 67, 611–616. [39] S. M. Ametamey, Chem. Rev. 2008, 108, 4036–4036. [40] P. W. Miller, N. J. Long, R. Vilar, A. D. Gee, Angew. Chem. Int. Edit. 2008, 47, 8998–9033. [41] G. N. Reddy, M. Haberli, H. F. Beer, A. P. Schubiger, Appl. Radiat. Isot. 1993, 44, 645–649. [42] A. Plenevaux, J. S. Fowler, S. L. Dewey, A. P. Wolf, M. Guillaume, Int. J. Rad. Appl. Instrum. A 1991, 42, 121–127. [43] S. Nag, L. Lehmann, G. Kettschau, T. Heinrich, A. Thiele, A. Varrone, B. Gulyas, C. Halldin, Bioorg. Med. Chem. 2012, 20, 3065–3071.

88 www.jlcr.org

Copyright © 2013 John Wiley & Sons, Ltd.

J. Label Compd. Radiopharm 2013, 56 78–88