Synthesis and Autoradiography of Novel F-18 Labeled Reversible Radioligands for Detection of Monoamine Oxidase B.

Monoamine oxidase B (MAO-B) is an important enzyme regulating the levels of monoaminergic neurotransmitters. Selective MAO-B inhibitors have been labeled with carbon-11 or fluorine-18 to visualize the localization of MAO-B in vivo by positron emission tomography (PET) and thereby have been useful for studying neurodegenerative diseases. The aim of this study was to develop promising fluorine-18 labeled reversible MAO-B PET radioligands and their biological evaluation in vitro by autoradiography. Radiolabeling was achieved by classical one-step fluorine-18 nucleophilic substitution reaction. The stability and radiochemical yield was analyzed with HPLC. All five fluorine-18 labeled compounds were tested in human whole hemisphere autoradiography experiments. Five compounds (GEH200439, GEH200448, GEH200449, GEH200431A, and GEH200431B) were successfully radiolabeled with fluorine-18, and the incorporation yield of the fluorination reactions varied from 10 to 45% depending on the compound. The radiochemical purity was higher than 99% for all at the end of synthesis. Radioligands were found to be stable, with a radiochemical purity of >99% in a sterile phosphate buffered saline (pH = 7.4) over the duration of the study. The ARG binding density of only 18F-GEH200449 was consistent with known MAO-B expression in the human brain. Radiolabeling of five new fluorine-18 MAO-B reversible inhibitors was successfully accomplished. Compound 18F-GEH200449 binds specifically to MAO-B in vitro postmortem brain and could be a potential candidate for in vivo PET investigation.

Introduction

Monoamine oxidase is an intracellular enzyme mounted in the outer membrane of mitochondria in neuronal and non-neuronal cells in the brain and in most cell types of peripheral organs.[1] The two isoforms of monoamine oxidase (MAO), referred to as “MAO Type A” (MAO-A) and “MAO Type B” (MAO-B), are differentiated according to biochemical and pharmacological properties.

In humans, MAO-A is mostly expressed in placenta, adipose tissue, thyroid gland, and lung, whereas its expression in the brain is low. In contrast, MAO-B is predominantly expressed in various parts of the CNS and has been estimated to constitute up to ∼70% of total brain MAO activity.[2] High level of MAO-B is primarily found in basal forebrain, brainstem, basal ganglia, and thalamus. Cerebral MAO-B level increases linearly in an age-dependent manner during normal aging and has been reported to be further upregulated in Alzheimer’s Disease (AD) patients.[3,4] Of further interest in research on the pathophysiology of neurodegenerative conditions is the observation that reactive astrocytes may also express MAO-B. Because of the enzymes’ central role in the neurotransmitter metabolism, MAO-B has since long been established as a target in the pharmacological therapy of psychiatric disorders such as depression[5] and social anxiety[6] as well as in Parkinson’s disease (PD).[7]

The broad interest in MAO-B is a reason for the wide use of the noninvasive imaging technique positron emission tomography (PET) to visualize the anatomical distribution of radioligand binding to MAO-B in the brain. For that purpose, a number of selective radioligands (irreversible and reversible) have been developed over the years.[8−10] Most of the radioligands[11−13] bind in an irreversible manner to MAO-B, whereas only 18F-Ro 43-0463,[14]11C-SL25.1188,[15] and 18F-FSL25.1188[16] have shown more reversible binding properties.

Among the radioligands, only carbon-11 labeled compounds such as 11C-deprenyl,[17]11C-L-deprenyl-D2,[18] and 11C-SL25.1188[19] have been validated and used clinically.[20] However, the short half-life of carbon-11 (20.4 min) also makes carbon-11 labeled radioligands less suitable for use at PET centers not having a cyclotron. Another drawback of 11C-deprenyl is that its main radiometabolite 11C-methamphetamine enters the brain. For those reasons, there is a need for radioligands labeled with fluorine-18 (half-life 110 min) that are useful for wider clinical research.

Recent developments on the structure–activity relationship suggests that coumarin, chromone, oxazolidinone, and benzoxazole derivatives are selective and reversible MAO-B inhibitors.[21,22] In this project, we selected five compounds GEH200439 (benzoxazole derivative), GEH200448 (coumarin derivative), GEH200449 (chromone derivative), GEH200431A, and GEH200431B (oxazolidinone derivative) (Figure ) for radiolabeling with fluorine-18. Our aims were to develop syntheses methods for all five compounds and to evaluate the binding properties in vitro by autoradiography of the postmortem human brain.

Figure 1

Structures of five compounds GEH200439 (benzoxazole derivative), GEH200448 (coumarin derivative), GEH200449 (chromone derivative), GEH200431A, and GEH200431B (oxazolidinone derivatives).

Results and Discussions

Radiochemistry

The radiolabeling was achieved by one-step nucleophilic substitution reaction of the corresponding precursor (GEH200438, GEH200451, GEH200452, GEH200454, and GEH200455) with 18F-fluoride in the presence of Kryptofix[2.2.2] (K2.2.2) and K2CO3 as shown in Figure . Azeotropic drying was performed before the dried K18F–F–K2.2.2 complex was treated with a specific amount of the corresponding precursor. Different solvents such as acetonitrile, DMF, and DMSO were tested at different temperatures. DMSO was found to be the best solvent for all radiolabeling syntheses. Therefore, the general use of DMSO as reaction solvent and specific reaction temperature and time for each of the different precursors (GEH200438, GEH200451, GEH200452, GEH200454, and GEH200455) resulted in the desired product with best radiochemical yield (Table ). The overall radiosynthesis including 18F-fluorination, HPLC purification, and SPE isolation followed by the formulation was completed within the time range 75–90 min.

Figure 2

Radiosynthesis of GEH200439, GEH200448, GEH200449, GEH200431-A, and GEH200431-B with fluorine-18.

Table 1

Optimization of Radiolabeling

entry	precursor (mg)	reaction time (min)	reaction temperature (oC)	RCY (%)	MA (GBq/μmol)
18F-GEH200439	2–3	10	135	47 ± 5	81 ± 55
					(n = 6)
18F-GEH200448	6	20	160	3	146
					(n = 2)
18F-GEH200449	2	10	125	16 ± 5	139 ± 29
					(n = 6)
18F-GEH200431A	3	15	120	23 ± 4	160 ± 101
					(n = 3)
18F-GEH200431B	3	15	120	21 ± 6	147 ± 67
					(n = 4)

The incorporation yield of the fluorination reactions varied from 3 to 52%, and the radiochemical purity was higher than 99% for all five compounds (Table ). The identities of the 18F labeled compounds were confirmed by coinjection of their corresponding fluorine-19 analogues of GEH200439, GEH200448, GEH200449 (Figure ), GEH200431A, and GEH200431B using analytical HPLC. All radioligands were found to be stable in PBS buffered saline (pH = 7.4) for the duration of 120 min with a half-life of 110 min.

Figure 3

(A) HPLC chromatogram of the semipreparative purification of 18F-GEH200449 and (B) HPLC chromatogram of the analysis of 18F-GEH200449 coinjected with the cold reference standard GEH200449.

Autoradiography

All five fluorine-18 labeled compounds, 18F-GEH200439, 18F-GEH200431A, 18F-GEH200431B, 18F-GEH200448, and 18F-GEH200449, were examined by human whole hemisphere autoradiography on brain tissue obtained from deceased subjects with no sign of any brain disorder. 18F-GEH200439 featured high binding to white matter and no binding to cortical gray matter (data not shown). The white matter binding could not be blocked with L-deprenyl. To exclude binding to other targets, we extended the protocol for 18F-GEH200439 and included self-inhibition with unlabeled GEH200439. There was no evident effect of self-inhibition indicating that the observed binding is mainly nonspecific. Similar to 18F-GEH200439, both 18F-GEH200431A and 18F-GEH200431B featured high binding to white matter, and the binding could not be blocked by an excess (10 μM) of L-deprenyl or the MAO-A ligand pirlindole (data not shown). It can be concluded that both 18F-GEH200431A and 18F-GEH200431B are not suitable as radioligands for detection of MAO-B.

The binding pattern of 18F-GEH200448 was different from that of the other radioligands. There was a conspicuous accumulation of radioactivity in hippocampus, putamen, caudate, cerebellum, and thalamus (Figure ). An excess (10 μM) of the MAO-B specific ligand L-deprenyl inhibited 40–50% of 18F-GEH200448 binding in the caudate, putamen, and globus pallidus and approximately 20% of 18F-GEH200448 binding in the cerebellum. In addition, the MAO-A specific ligand pirlindole (10 μM) inhibited approximately 10–15% of 18F-GEH200448 binding in the caudate, putamen, and globus pallidus as well as in the cerebellum (Figure ). These observations indicate that 18F-GEH200448 displays specific binding in MAO-B rich brain structures and shows moderately high nonspecific binding.

Figure 4

Autoradiograms of horizontal slices of a human brain labeled with 18F-GEH200448 at baseline condition and during incubation with L-deprenyl (10 μM) and pirlindole (10 μM).

Finally, the binding of 18F-GEH200449 was high in all MAO-B-containing regions, and the signal intensities were highest in the hippocampus, putamen, caudate, and thalamus. The MAO-B specific ligand L-deprenyl (10 μM) completely inhibited 18F-GEH200449 binding in all regions, whereas the MAO-A specific ligand pirlindole (10 μM) blocked less than 10% of the total binding (Figure ). These observations indicate that 18F-GEH200449 binds specifically and selectively to MAO-B in the human brain. Moreover, the contrast between specific and nonspecific binding (background) appears to be high.

Figure 5

Autoradiograms of horizontal slices of a human brain labeled with 18F-GEH200449 at baseline condition and during incubation with L-deprenyl (10 μM) and pirlindole (10 μM).

Conclusions

The present study demonstrated that the five potential MAO-B radioligands could efficiently be labeled with fluorine-18. Only 18F-GEH200449 exhibited high specific binding to MAO-B in the postmortem human brain autoradiography. The results suggest that 18F-GEH200449 has potential for further development as a PET radioligand for imaging of binding to MAO-B in the human brain in vivo.

Materials and Methods

General

All the precursors (GEH200438, GEH200451, GEH200452, GEH200454, and GEH200455) and all the nonradioactive reference standards (GEH200439, GEH200448, GEH200449, GEH200431A, and GEH200431B) were supplied by GE Healthcare. All other chemicals and reagents were obtained from commercial sources and used without any further purification. Solid-phase extraction (SPE) cartridges SepPak QMA light and SepPak C18 Plus were purchased from Waters (Milford, MA, USA). C18 Plus cartridge was activated using EtOH (10 mL) and followed by sterile water (10 mL). SPE cartridge SepPak QMA light was activated using K2CO3 solution (0.5M, 10 mL) and followed by water (15 mL, 18 MΩ,). Fluorine-18 fluoride was produced at the Karolinska Hospital (Stockholm, Sweden). Radiolabeling was performed using a custom-made semiautomated synthesis module. Liquid chromatographic analysis (LC) was performed with a Merck-Hitachi gradient pump and a Merck-Hitachi, L-4000 variable wavelength UV detector. LC–MS was performed using a Waters Quattra-Tof Premier micro mass spectrometer, or Waters SQD 3001 single quadrupole mass spectrometer, coupled to Waters Acquity UPLC instruments.

Production of 18F-Fluoride (18F–F–)

Fluorine-18 fluoride (18F–F) was produced from a GEMS PETtrace Cyclotron using 16.4 MeV protons via the 18O(p,n)18F reaction on 18O enriched water ([18O]H2O). [18F–F was isolated from 18O–H2O on a preconditioned SepPak QMA light anion exchange cartridge and subsequently eluted from the cartridge with a solution of K2CO3 (13 μmol, 1.8 mg), Kryptofix 2.2.2 (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo-[8.8.8]hexacosane-K2.2.2) (26 μmol, 9.8 mg) in water (85 μL, 18 MΩ,) and MeCN (2 mL) to a reaction vessel (10 or 4 mL). The solvents were evaporated at 140 °C for 10–15 min under continuous nitrogen/helium flow (70 mL/min) to form a dry complex of 18F–F/K2CO3/K2.2.2, and the residue was cooled to room temperature (RT).

Synthesis of 18F-GEH200439 (5-18F-2-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)oxazolo[5,4-b]pyridine)

To the dry complex of 18F–F–/K2CO3/K2.2.2, corresponding precursor (510min-chloro-2-(1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)oxazolo[5,4-b]pyridine) (2–3 mg, 0.007–0.011 mmol) in DMSO (700 μL) was added at 135 °C and left for 10 min to produce 18F-GEH200439. The reaction mixture was cooled to RT and was diluted with water to a total volume of 3 mL before it was injected into a semipreparative reverse phase ACE C-18 HPLC column (C18, 7.8 Ø × 250 mm, 5 μm) for purification. The column outlet was connected to a UV absorbance detector (λ = 254 nm) in series with a GM tube for radioactivity detection. Elution with mobile phase CH3CN/amoniumformate (0.1M) (60/40) at a flow rate of 5 mL/min gave a radioactive fraction corresponding to pure 18F-GEH200439 with a retention time (tR) of 18–20 min.

Synthesis of 18F-GEH200448 (3-(2-18F-Fluoropyridin-4-yl)-6-methyl-2H-chromen-2-one)

To the dry complex of 18F–F–/K2CO3/K2.2.2, corresponding precursor (3-(2-chloropyridin-4-yl)-6-methyl-2H-chromen-2-one) (6 mg, 0.023 mmol) in DMSO (500 μL) was added at 160 °C and left for 20 min to produce 18F-GEH200448. The reaction mixture was cooled to RT and was diluted with water to a total volume of 3 mL before it was injected into a semipreparative reverse phase ACE C-18 HPLC column (C18, 7.8 Ø × 250 mm, 5 μm) for purification. The column outlet was connected to a UV absorbance detector (λ = 254 nm) in series with a GM tube for radioactivity detection. Elution with mobile phase CH3CN/TFA (0.1%) (45/55) at a flow rate of 6 mL/min gave a radioactive fraction corresponding to pure 18F-GEH200448 with a retention time (tR) of 14–16 min.

Synthesis of 18F-GEH200449 (6-((6-18F-Fluoropyridin-3-yl)methoxy)-4H-chromen-4-one)

To the dry complex of 18F–F–/K2CO3/K2.2.2, corresponding precursor (6-((6-chloropyridin-3-yl)methoxy)-4H-chromen-4-one) (2 mg, 0.007 mmol) in DMSO (600 μL) was added at 125 °C and left for 10 min to produce 18F-GEH200449. The reaction mixture was cooled to RT and was diluted with water to a total volume of 3 mL before it was injected into a semipreparative reverse phase ACE C-18 HPLC column (C18, 7.8 Ø × 250 mm, 5 μm) for purification. The column outlet was connected to a UV absorbance detector (λ = 254 nm) in series with a GM tube for radioactivity detection. Elution with mobile phase CH3CN/TFA (0.1%) (30/70) at a flow rate of 6 mL/min gave a radioactive fraction corresponding to pure 18F-GEH200448 with a retention time (tR) of 18–19 min.

Synthesis of 18F-GEH200431-A ((S)-3-(6-((R)-2-18F-Fluoro-4,4,4-trifluorobutoxy)benzo[d]isoxazol-3-yl)-5-(methoxymethyl)oxazolidin-2-one) and 18F-GEH200431-B (((S)-3-(6-((S)-2-18F-Fluoro-4,4,4-trifluorobutoxy)benzo[d]isoxazol-3-yl)-5-(methoxymethyl)oxazolidin-2-one

To the dry complex of 18F–F–/K2CO3/K2.2.2, corresponding precursor GEH200454 ((S)-4,4,4-trifluoro-1-((3-((S)-5-(methoxymethyl)-2-oxooxazolidin-3-yl)benzo[d]isoxazol-6-yl)oxy)butan-2-yl-4-methylbenzenesulfonate) or GEH200455 ((R)-4,4,4-trifluoro-1-((3-((S)-5-(methoxymethyl)-2-oxooxazolidin-3-yl)benzo[d]isoxazol-6-yl)oxy)butan-2-yl-4-methylbenzenesulfonate) (3 mg, 0.005 mmol) in DMSO (500 μL) was added at 120 °C and left for 15 min to produce 18F-GEH200431-A or 18F-GEH200431-B. The reaction mixture was cooled to RT and was diluted with water to a total volume of 3 mL before it was injected into a semipreparative reverse phase ACE C-18 HPLC column (C18, 7.8 Ø × 250 mm, 5 μm) for purification. The column outlet was connected to a UV absorbance detector (λ = 254 nm) in series with a GM tube for radioactivity detection. Elution with mobile phase CH3CN/amoniumformate (0.1M) (60/40) at a flow rate of 5 mL/min gave a radioactive fraction corresponding to pure 18F-GEH200448 with a retention time (tR) of 23–25 min.

Isolation of 18F-GEH200439, 18F-GEH200448, 18F-GEH200449, 18F-GEH200431-A, and 18F-GEH200431-B

The corresponding radioactive fraction collected from HPLC was diluted with sterile water (50 mL). The resulting mixture was loaded onto a preconditioned (10 mL of ethanol followed by 10 mL of sterile water) SepPak tC18 plus cartridge. The cartridge was washed with sterile water (10 mL), and the corresponding isolated 18F-product was eluted with 1 mL of ethanol into a sterile vial containing phosphate buffered saline solution (PBS, 9 mL).

Quality Control and Molar Activity (MA) Determination

The radiochemical purity, identity, and stability of 18F-GEH200439, 18F-GEH200448, 18F-GEH200449, 18F-GEH200431A, and 18F-GEH200431B were determined by the analytical HPLC system which included an ACE RP column (C18, 3.9 Ø × 250 mm, 5 μm particle size), Merck-Hitatchi L-7100 Pump, L-7400 UV detector, and GM tube for radioactivity detection (VWR International). The mobile phase CH3CN/0.1% TFA with a gradient HPLC method (15–90% in 10 min) and flow rate of 2 mL/min was used to elute the product. The effluent was monitored with a UV absorbance detector (ƛ = 254 nm) coupled to a radioactive detector (β-flow, Beckman, Fullerton, CA). The identity of fluorine-18 labeled compounds was confirmed by using HPLC with the coinjection of the corresponding authentic nonradioactive reference standard.

The MA of the final product was measured by analytical HPLC which included an ACE RP column (C18, 3.9 Ø × 250 mm, 5 μm particle size) using mobile phase CH3CN/50 mM H3PO4 with a gradient HPLC method (10–90% in 10 min) and flow rate of 2 mL/min. MA was calibrated for UV absorbance (ƛ = 254 nm) response per mass of ligand and calculated as the radioactivity of the radioligand (GBq) divided by the amount of the associated carrier substance (μmol). Each sample was analyzed three times and compared to a reference standard also analyzed three times.

In Vitro Autoradiography

Human brains without pathology were obtained from the National Institute of Forensic Medicine, Karolinska Institutet (Stockholm, Sweden). The brains had been removed during forensic autopsy (control brains) and were handled in a manner similar to that described previously.[23−25]

In the present study, tissue was obtained from a 32-year-old male donor, and the postmortem time was 12 h. After the removal of the brain, it was kept at −85 °C until sectioning, after which the whole hemisphere brain slices were kept at −25 °C until the autoradiography procedures. Ethical permissions were obtained from the Ethics Committee at Karolinska Institutet (registration no. 03-767). The sectioning of the brains and the autoradiography experiments were performed at the Department of Neuroscience, Karolinska Institutet. Whole brain hemispheres were sectioned into 100 μm thick horizontal slices using a Leica cryomacrocut system. The autoradiographic procedures were identical with the former studies done in our laboratory.[26,27] Briefly, 100 μm thick whole hemisphere sections were incubated for 90 min at room temperature with 4 MBq (megabecquerel)/200 mL of the corresponding radiotracer in 50 mM TRIS buffer pH 7.4 containing sodium chloride (120 mM), potassium chloride (5 mM), calcium chloride (2 mM), and albumin (0.1% w/v). After the incubation, the sections were washed in the same buffer three times for 5 min each time at room temperature, briefly dipped in ice cold distilled water, dried, and exposed to phosphorimaging plates. Standards for quantification of the binding density were prepared by serial dilution of the radioligand stock solution in assay buffer. The readings were made in a Fujifilm BAS-5000 phosphorimager, using the phosphorimager’s Multi Gauge 3.2 image analysis software (Fujifilm) for quantitative analysis. Blocking experiments were performed by coincubating adjacent brain sections with L-deprenyl (10 μmol), pirlindole (10 μM), or GEH200439 (10 μM).