Elsevier

Bioorganic & Medicinal Chemistry

Volume 21, Issue 21, 1 November 2013, Pages 6634-6641
Bioorganic & Medicinal Chemistry

Development of a novel fluorine-18 labeled deuterated fluororasagiline ([18F]fluororasagiline-D2) radioligand for PET studies of monoamino oxidase B (MAO-B)

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Abstract

The objective of this study was to synthesize and evaluate a novel fluorine-18 labeled deuterium substituted analogue of rasagiline (9, [18F]fluororasagiline-D2) as a potential PET radioligand for studies of monoamine oxidase B (MAO-B).

The precursor compound (6) and reference standard (7) were synthesized in multi-step syntheses. Radiolabeling of 9 was accomplished by a two-step synthesis, compromising a nucleophilic substitution followed by hydrolysis of the sulfamidate group. The incorporation radiochemical yield from fluorine-18 fluoride was higher than 30%, the radiochemical purity was >99% and the specific radioactivity was >160 GBq/μmol at the time of administration.

In vitro compound 7 inhibited the MAO-B activity with an IC50 of 173.0 ± 13.6 nM. The MAO-A activity was inhibited with an IC50 of 9.9 ± 1.1 μM. The fluorine-18 version 9 was characterized in the cynomolgus monkey brain where a high brain uptake was found (275% SUV at 4 min). There was a higher uptake in the striatum and thalamus compared to the cortex and cerebellum. A pronounced blocking effect (50% decrease) was observed in the specific brain regions after administration of l-deprenyl (0.5 mg/kg) 30 min prior to the administration of 9. Radiometabolite studies demonstrated 40% of unchanged radioligand at 90 min post injection.

An efficient radiolabeling of 9 was successfully established and in the monkey brain 9 binds to MAO-B rich regions and its binding is blocked by the selective MAO-B compound l-deprenyl. The radioligand 9 is a potential candidate for human PET studies.

Graphical abstract

Fluorine-18 labeled deuterated analogue of fluororasagiline for detection of MAO-B activity by PET.

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Introduction

Flavin adenine dinucleotide (FAD) dependent enzymes monoamine oxidases A and B (MAO-A and MAO-B) are responsible for the metabolism of neurotransmitters such as dopamine, serotonin, adrenaline, and noradrenaline.1 Both enzymes are bound to the outer mitochondrial membrane and differ in their distribution in body organs and in their substrate specificity as well as exhibit different inhibitor specificities. MAO-B is the predominant isoform of MAO enzymes in human brain and primarily located in astrocytes and serotonergic neurons in the normal as well as the lesioned brain.2 It metabolizes dopamine to 3,4-dihydroxyphenylacetic acid and homovanillic acid, as well as is responsible for the deamination of β-phenylethylamine, an endogenous amine that stimulates the release and inhibits neuronal reuptake of dopamine.3 Because of its central role in neurotransmitters metabolism, MAO-B represents an attractive drug targets for the treatment of diseases such as AD,4,5 PD6 and depression.7 MAO-B is selectively and irreversibly inhibited by such as l-deprenyl and rasagiline which are co-administrated for the treatment of relieving symptoms resulting from the loss of dopaminergic neurons in Parkinson’s disease.8 Eventhough both rasagiline and l-deprenyl (also known as Selegiline) feature a propargyl group; there are significant differences in their base structures and metabolic products. The main metabolite of l-deprenyl is l-methamphetamine which is further metabolized to l-amphetamine, has neurotoxic properties in experimental models.9 This amphetamine action may contribute towards to side effects such as increased heart rate and blood pressure.10 In contrast, rasagiline does not have any neurotoxic metabolite. Its main metabolite is R-aminoindan, which does not increase noradrenaline potency as well as does not display any MAO-B inhibitory activity.11

Positron emission tomography (PET), a high-resolution, sensitive and non-invasive imaging technique has been widely utilized in visualizing the localization of MAO-B.12 Imaging brain MAO-B activity with PET in humans has been useful for studying neurodegenerative diseases13, 14 and epilepsy.15 In our previous publications we have reported several fluorine-18 labeled radio ligands such as [18F]fluorodeprenyl,16, 17 [18F]fluororasagiline, 18 (S)-N-(1-[18F]fluoro-3-(furan-2-yl)propan-2-yl)-N-methylprop-2-yn-1-amine and (S)-1-[18F]fluoro-N,4-dimethyl-N-(prop-2-ynyl)pentan-2-amine19 for study of MAO-B. The present study is the continuation of our previous work where we reported the synthesis and in vivo evaluation of MAO-B in cynomolgus monkey brain using [18F]fluororasagiline as a PET radioligand and also investigated its radiometabolism in blood plasma.18 We concluded that a continuing increase of the radioactivity throughout the PET scan may indicate a blood–brain barrier penetrating radiometabolite that may in turn complicate a reliable quantification. This could be explained by the distribution of [18F]fluororasagilinein tissue limited by blood flow rather than by the MAO-B enzyme concentration in regions with high MAO-B activity as it was shown for [11C]deprenyl.20

Kinetic isotope effect has widely been used in the elucidation of organic as well as enzymatic reaction mechanism.21 The deuterium isotope effect has an important role in the oxidative deamination of amines by MAO.22 Thus the MAO-B catalyzed bond cleavage of carbon–hydrogen bond of propargyl group at α carbon is the rate limiting step in the retention of radioligand in the brain.23 In our recent publication we showed that the deuterium substituted analogue of [18F]fluorodeprenyl reduced the rate of radioligand trapping in monkey brain and improved its sensitivity as well as reduced the metabolism in plasma (Fig. 1).24

Therefore the objective of the present study was threefold: (i) to prepare the precursors and reference standards and to develop efficient synthetic methods for labeling deuterated fluororasagiline with fluorine-18 (ii) to characterize it is in vitro MAO-B and MAO-A inhibition based on the rate of kynuramine oxidation and transformation to 4-OH quinoline generation, and (iii) to evaluate the in vivo characteristics by PET measurement in a non-human primate. The overall aim was to evaluate the radioligand as a prospective PET radioligand for imaging MAO-B

Section snippets

Chemistry

The precursor compound (6) and the reference standard (7) were synthesized by multistep organic syntheses (Scheme 1). In the first part of the synthesis (Scheme 1) compound 3 was prepared following a previously described method where the commercially available compound propiolic acid (1) was treated with lithium aluminum deuteride to synthesise (1,1-2H2)prop-2-yn-1-ol (2). The key parameter of this reaction was to keep the temperature at below −50 °C. The synthesized compound (1,1-2H2

Conclusions

Radiolabelling of a deuterium substituted analogue of fluororasagiline (9) was accomplished with a sufficient radiochemical yield for further application in PET. In vivo characteristics in cynomolgus monkey showed a high brain uptake of 9 in known MAO-B rich regions. The deuterated analogue of fluororasagiline (9) measured in monkey plasma is more stable compared to the nondeuterated analogue. These results together suggest that 9 may be an improved PET radioligand and potential molecular

Chemistry

NMR spectra were recorded on Varian Unity-400 and Bruker Avance 400 (1H, 400 MHz and 13C, 100 MHz), and Bruker Avance 600 III (1H, 600 MHz) NMR instruments. 1H NMR spectra were referenced internally on CDCl3 (δ 1H 7.26) and 13C NMR spectra were referenced internally on CDCl3 (δ 13C 77.20). All chemicals and solvents were purchased from Sigma–Aldrich and used without any further purification unless otherwise stated. Dry acetonitrile (MeCN; max 10 ppm H2O) was purchased from Merck. Solid-phase

Acknowledgments

The authors would like to thank all the members of Karolinska Institutet PET centre for assistance in the PET experiments including special thanks to Gudrun Nylen, Jonas Bergstrom, and Guennadi Jogolev for excellent technical assistance and to Bayer HealthCare AG for their support. The research leading to these results has also received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n° HEALTHF2-2011-278850 (INMiND). The compound [18

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