Development of N-[3-(2′,4′-dichlorophenoxy)-2-18F-fluoropropyl]-N-methylpropargylamine (18F-fluoroclorgyline) as a potential PET radiotracer for monoamine oxidase-A

July 9, 2017 | Autor: Jogeshwar Mukherjee | Categoria: Positron Emission Tomography, Rat Brain, Clinical Sciences, Specific Activity, Monoamine oxidase
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Nuclear Medicine & Biology, Vol. 26, pp. 619 – 625, 1999 Copyright © 1999 Elsevier Science Inc. All rights reserved.

ISSN 0969-8051/99/$–see front matter PII S0969-8051(99)00027-X

Development of N-[3-(2⬘,4⬘-Dichlorophenoxy)-2-18FFluoropropyl]-N-Methylpropargylamine (18F-Fluoroclorgyline) as a Potential PET Radiotracer for Monoamine Oxidase-A Jogeshwar Mukherjee and Zhi-Ying Yang FRANKLIN MCLEAN INSTITUTE, DEPARTMENT OF RADIOLOGY AND DEPARTMENT OF PHARMACOLOGICAL AND PHYSIOLOGICAL SCIENCES, UNIVERSITY OF CHICAGO, CHICAGO, ILLINOIS, USA

ABSTRACT. We have synthesized N-[3-(2ⴕ,4ⴕ-dichlorophenoxy)-2-18F-fluoropropyl]-N-methylpropargylamine (18F-fluoroclorgyline) as a potential positron emission tomography (PET) radiotracer for monoamine oxidase A (MAO-A). The radiosynthesis was carried out by a 18F-fluoride-for-mesylate substitution reaction in approximately 20% radiochemical yield in specific activities of 1–2 Ci/␮mol. Selectivity for MAO-A was demonstrated by the high affinity of clorgyline (IC50 ⴝ 39 nM) and lower affinity of (R)-deprenyl (IC50 > 100 ␮M) for the inhibition of 18F-fluoroclorgyline binding in vitro in rat brain membranes. The uptake of 18F-fluoroclorgyline in the rat brains was high (>1.0% injected dose/g). The binding of 18F-fluoroclorgyline in the rat brain correlated with the distribution of MAO-A and was inhibited by preadministration of MAO-A inhibitors, clorgyline, and Ro 41-1049, whereas (R)-deprenyl, a MAO-B blocker, had no inhibitory effect. These results suggest that 18F-fluoroclorgyline is a potential PET radiotracer for MAO-A. NUCL MED BIOL 26;6:619 – 625, 1999. © 1999 Elsevier Science Inc. All rights reserved. KEY WORDS.

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F-Fluoroclorgyline, Monoamine oxidase A , Fluorine-18

INTRODUCTION Monoamine oxidase A (MAO-A) has been investigated noninvasively by using radioligands developed for positron emission tomography (PET) and single photon emission computed tomography (SPECT). Carbon-11-labeled irreversible radiotracers, such as 11Cclorgyline has been extensively studied in humans (3, 8). Efforts have also been made to develop iodinated and fluorinated analogs of clorgyline, which may provide potential radioligands for the study of this enzyme system (Fig. 1). Other radiotracers for MAO-A, such as 11C-harmine and derivatives and 11C-brofaromine (1, 2), have also been investigated more recently for use in PET. Our interest was to develop a fluorine-18 radiotracer for the study of MAO-A to use in the evaluation of various antidepressants for their affinity to the enzyme in vitro and their ability to inhibit the enzyme in vivo. The longer half-life (110 min) of fluorine-18 would allow the in vitro and in vivo pharmacological evaluation of the various drugs over an extended period of time compared with carbon-11 (half-life of 20 min). A brief structure–activity relationship of clorgyline has been carried out with iodine, fluorine and phenoxy substitutions (11, 13, 14). A fluorine-18-labeled analog of clorgyline, using the lower specific activity electrophilic fluorine18, has been reported previously and shown to localize in MAO-A sites in rodents (5). We decided on the development of irreversible agents due to the somewhat lesser synthetic and radiosynthetic difficulties posed by these compounds. There are only limited structure–activity relationship data available on fluorinated derivatives of clorgyline (13). Address correspondence to: Jogeshwar Mukherjee, Ph.D., Department of Nuclear Medicine/PET, Kettering Medical Center, 3535 Southern Boulevard, Dayton, OH 45429, USA; e-mail: [email protected] Received 8 August 1998. Accepted 5 April 1999.

A 11C-radiolabeled tracer of an aromatic ring fluorinated derivative showed promise in localizing to MAO-A sites in mice (14). A preliminary report of a low specific activity electrophilic fluorine18-labeled clorgyline derivative with promising properties has been reported (5). Due to the general difficulty of radiolabeling with nucleophilic fluorine-18 on electron-rich aromatic rings in order to obtain a high specific activity fluorine-18 radiotracer, we investigated other sites for incorporating the fluorine atom. We envisioned placing the fluorine on the propyl group, at the ␤-carbon of clorgyline, which would potentially provide a fluorinated MAO-A inhibitor that could be developed into a 18F-fluorinated derivative as a potential PET radiotracer. We report here the radiosynthesis and in vitro and in vivo characterization of N-[3-(2⬘,4⬘dichlorophenoxy)-2-18F-fluoropropyl]-N-methylpropargylamine (18F-fluoroclorgyline) as a potential PET radiotracer for MAO-A. MATERIALS AND METHODS N-Methylpropargylamine, 2,4-dichlorophenol, and 1,3-dibromo-2propanol were purchased from Aldrich Chemical Co. (Milwaukee, WI) and clorgyline, Ro 41-1049 (N-(2-aminoethyl)-5-(3-fluorophenyl)-4-thiazolecarboxamide) and (R)-deprenyl were purchased from Research Biochemicals Inc. (Natick, MA). All other reagents were of analytical grade and were used without further purification. Analytical and preparative thin-layer chromatography (TLC) were performed using Baker-flex silica gel IB-F and Alltech DC-Fertigplatten SIL G-200 UV254 plates, respectively. High pressure liquid chromatography (HPLC) was carried out on a Gilson Gradient System consisting of two Gilson pumps and one ultraviolet (UV) detector with wavelength fixed at 254 nm and a radiation flow detector with a NaI(Tl) crystal. Solvent A of the gradient was 0.01 M phosphoric acid, pH 4.7, and solvent B was acetonitrile. Semi-prep (250 ⫻ 10 mm) C18 columns from Alltech Associates

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FIG. 1. Chemical structures of radiolabeled clorgyline analogs as monoamine oxidase A inhibitors.

Inc. (Deerfield, IL) were used for reverse-phase HPLC. Proton nuclear magnetic resonance (NMR) spectra were obtained on a GE NMR OMEGA 500 MHz spectrometer, and electron-impact mass spectra were obtained on a VG Instruments Inc. Model 7250 mass spectrometer.

Synthesis 3-(2ⴕ,4ⴕ-DICHLOROPHENOXY)-2-HYDROXYPROPYL BROMIDE (1). A round-bottomed flask was charged with 2,4-dichlorophenol (0.16 g;

1 mmol) and dissolved in 1 mL of dimethylacetamide (DMA). Into this solution was added sodium hydride (0.05 g; 2 mmol), and the solution was heated at 50°C for 30 min. Into this reaction mixture, 1,3-dibromo-2-propanol (0.44 g; 2 mmol) was added and the reaction was heated overnight (20 h) at 70°C (reaction scheme shown in Fig. 2) and the product appeared with an Rf ⫽ 0.6 (3:1 hexane-ethylacetate). The reaction mixture was subsequently quenched with water and the product was extracted with ether and purified on preparative TLC (3:1 hexane-ethylacetate) to provide 3-(2⬘,4⬘-dichlorophenoxy)-2-hydroxypropyl bromide in 45% yield.

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F-Fluoroclorgyline as a Radiotracer for Monoamine Oxidase A

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FIG. 2. Synthesis scheme of the mesylate precursor 3 (N-[3-(2ⴕ,4ⴕ-dichlorophenoxy)-2-mesyloxypropyl]-N-methylpropargylamine, N-[3-(2ⴕ,4ⴕ-dichlorophenoxy)-2-fluoropropyl]-N-methylpropargylamine 4 (fluoroclorgyline), and N-[3-(2ⴕ,4ⴕ-dichlorophenoxy)-2-18F-fluoropropyl]-N-methylpro- pargylamine 18F-4 (18F-fluoroclorgyline). NMR ␦ ppm: 7.37 (d, 1H, Ar-3H), 7.16 (dd, 1H, Ar-5H), 6.94 (d, 1H, Ar-6H), 4.05 (m, 1H, CH), 3.58 (d, 2H, OCH2), 3.42 (d, 2H, CH2Br). N-[3-(2ⴕ,4ⴕ-DICHLOROPHENOXY)-2-HYDROXYPROPYL]-N-

Alkylation was carried out by using the reported general method of alkylation of propargylamines (12, 17). In a round-bottomed flask, N-methylpropargylamine (69 mg, 1 mmol) was taken along with 3-(2⬘,4⬘-dichlorophenoxy)-2-hydroxypropyl bromide (0.30 g, 1 mmol) in triethylamine (100 ␮L) and this mixture was refluxed for 2 h. The product appeared with an Rf of 0.35 (5% CH3OH, 95% CH2Cl2). The mixture was purified on preparative TLC to provide the pure product, N-[3-(2⬘,4⬘dichlorophenoxy)-2-hydroxypropyl]-N-methylpropargylamine in 60% yield and used subsequently for mesylation. NMR ␦ ppm: 7.28 (d, 1H, Ar-3H), 7.10 (dd, 1H, Ar-5H), 6.8 (d, 1H, Ar-6H), 4.08 (d, 2H, OCH2), 3.72 (s, 2H, -NCH2CCH), 2.78 (s, 3H, NCH3). METHYLPROPARGYLAMINE (2).

N-[3-(2ⴕ,4ⴕ-DICHLOROPHENOXY)-2-MESYLOXYPROPYL]-N-

The alcohol, N-[3-(2⬘,4⬘-dichlorophenoxy)-2-hydroxypropyl]-N-methylpropargylamine (29 mg; 0.1 mmol) was taken in dichloromethane (2 mL) and pyridine (10 ␮L) and the mixture was cooled in an ice-water bath. Into this cooled

METHYLPROPARGYLAMINE (3).

solution was added methanesulfonyl chloride (10 ␮L, 0.1 mmol) and the reaction was stirred for 2 h. The product appeared with an Rf of 0.80 (5% CH3OH, 95% CH2Cl2). The reaction mixture was dried in vacuo, taken up in dichloromethane, and washed with water. The dried organic layer was subsequently purified on preparative TLC to provide the pure product, N-[3-(2⬘,4⬘-dichlorophenoxy)-2-mesyloxypropyl]-N-methylpropargylamine in 55% yield. NMR ␦ ppm: 7.35 (d, 1H, Ar-3H), 7.18 (dd, 1H, Ar-5H), 7.0 (d, 1H, Ar-6H), 4.04 (d, 2H, OCH2), 3.76 (s, 2H, -NCH2CCH), 3.62 (s, 3H, CH3SO2), 2.73 (s, 3H, NCH3); Mass spectra (m/z, %) 366 (M⫹, 4%). N-[3-(2ⴕ,4ⴕ-DICHLOROPHENOXY)-2-FLUOROPROPYL]-N-METHYLPROPAR-

The mesylate, N-[3-(2⬘,4⬘-dichlorophenoxy)-2-mesyloxypropyl]-N-methylpropargylamine, 3 (36 mg; 0.1 mmol) was taken in tetrahydrofuran (0.2 mL). Into this mixture was added tetrabutylammonium fluoride (TBAF; 0.1 mL of a 1-M tetrahydrofuran solution) and the reaction was refluxed for 45 min. The reaction mixture was subsequently taken to dryness, the residue taken up in CH2Cl2 and washed with water and dried over anhydrous magnesium sulfate. The product appeared with an Rf of 0.45 (5% CH3OH, 95% CH2Cl2). The dried organic layer was

GYLAMINE (4).

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FIG. 3. High performance liquid chromatography (HPLC) chromatogram of N -[3-(2ⴕ,4ⴕ-dichlorophenoxy)-2- 18 Ffluoropropyl]- N - methylpropargylamine (18F-fluoroclorgyline). The x-axis is time in minutes and y-axis is absorbance at 254 nm. Broken line is the radioactivity detector and the solid line is absorbance.

subsequently purified on preparative TLC to provide the pure product, N-[3-(2⬘,4⬘-dichlorophenoxy)-2-fluoropropyl]-N-methylpropargylamine in 30% yield. NMR ␦ ppm: 7.38 (d, 1H, Ar-3H), 7.12 (dd, 1H, Ar-5H), 6.90 (d, 1H, Ar-6H), 4.10 (d, 2H, OCH2), 3.46 (s, 2H, -NCH2CCH), 2.80 (s, 3H, NCH3); Mass spectra (m/z, %) 290 (M⫹, 8%). N-[3-(2ⴕ,4ⴕ-DICHLOROPHENOXY)-2-18F-FLUOROPROPYL]-N18 F-4). Aqueous F-fluoride (10 –100 mCi) was treated with potassium carbonate (0.34 mg) and Kryptofix (1.88 mg) in a Pyrex round-bottomed flask. The mixture was dried by heating at 85–90°C under a stream of nitrogen. Last traces of water were removed azeotropically with acetonitrile (1–2 mL). Into this dried Kryptofix-K18F complex, a solution (2 mg in 200 ␮L of acetonitrile) of N-[3-(2⬘,4⬘-dichlorophenoxy)-2-mesyloxypropyl]-N-methylpropargylamine was added, and this mixture was refluxed at 80 – 85°C. After 20 min, the acetonitrile in the mixture was dried under nitrogen and the residue was taken up in methanol. This methanolic solution was passed through a mini-column of neutral alumina, followed by washing the alumina with methanol. The methanolic eluate was evaporated to near dryness and the residue taken up in 60% methanol/40% buffer (⬍1 mL) and injected into the HPLC. Separation was carried out on an Alltech C18 column, using gradient elution with 0.01 M phosphoric acid and acetonitrile (95% 0.01 M phosphoric acid and 5% acetonitrile at 0 min, flow rate of 3 mL/min to 5% 0.01 M phosphoric acid and 60% acetonitrile at 20 min, flow rate of 3 mL/min). The retention time of the radiotracer, N-[3-(2⬘,4⬘dichlorophenoxy)-2-18F-fluoropropyl]-N-methylpropargylamine (18F-fluoroclorgyline) was 16.2 min, whereas that of the mesylate precursor was approximately 17.5 min (Fig. 3). The yields varied between 15% and 20% (decay-corrected) in several experiments with a radiochemical purity ⬎95%. To determine specific activity of 18F-fluoroclorgyline, the fraction collected between 15.6 and 16.5 min (containing the majority of the radioactive peak eluting at 16.2 min, as seen in Fig. 3) in the first HPLC purification was reinjected under the same gradient conditions for a second purification. A fraction between 15.6 and 16.5 min was collected to provide 18F-fluoroclorgyline and all the mass associated with this peak was assumed to be that of 18Ffluoroclorgyline for purposes of computing specific activity. This mass peak was compared with standard fluoroclorgyline injections.

METHYLPROPARGYLAMINE (18F-FLUOROCLORGYLINE, 18

The specific activity of HPLC purified between 1 and 2 Ci/␮mol.

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F-fluoroclorgyline varied

In Vitro Binding Assays Rat brains were isolated and homogenized with a Tekmar Tissumizer (15 s at half-maximum speed) in a 100-fold (w:v) dilution of a 50 mM Tris-HCl buffer, pH 7.4, containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM Na-ethylene diamine tetraacetic acid (Na-EDTA), and 0.1 mM Na ascorbate. The homogenate was centrifuged at 12,000 ⫻ g for 15 min at 4°C. The pellet was resuspended in the same volume of buffer, centrifuged a second time, and resuspended in fresh buffer at a concentration of 50 mg of tissue/mL. Each assay tube contained 0.10 mL of this stock solution. In vitro binding affinities of clorgyline, Ro 41-1049 and (R)deprenyl to MAO-A in rat (Sprague–Dawley) brain homogenates was carried out by incubating various concentrations (0.01 nM to 0.1 mM) of compounds along with the radioligand, 18F-fluoroclorgyline (specific activity 1 Ci/␮mol). Binding was initiated by addition of the tissue homogenate, and the tubes were incubated for 1 h at 37°C. The binding was terminated by filtration using a Brandel filtration apparatus, followed by washings with cold 50 mM Tris-HCl buffer (4 ⫻ 1 mL). Nonspecific binding was determined in the presence of 10 ␮M clorgyline. The filters were counted in a well-counter for fluorine-18 activity. The data were analyzed using the Ligand program and IC50 values for the various drugs were obtained (9). The binding curves were displayed using GraFit.

In Vivo Studies For in vivo studies, groups (n ⫽ 4) of Sprague–Dawley rats (250 g) were administered clorgyline (10 mg/kg), Ro 41-1049 (10 mg/kg), or (R)-deprenyl (10 mg/kg). All compounds (including saline, for control rats) were administered intraperitoneally under anesthesia, 90 min before injection of the radioligand and the rats were allowed free access to food and water during the interval. The radioligand, 18 F-fluoroclorgyline, 100 ␮Ci (specific activity 1 Ci/␮mol), was administered intravenously into each rat under anesthesia. The rats were subsequently allowed to recover and had free access to food and water. All rats were sacrificed 75 min after the radioligand injection and the various brain regions (cerebrum and cerebellum)

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F-Fluoroclorgyline as a Radiotracer for Monoamine Oxidase A

and blood were isolated into tared vials and counted for fluorine-18 activity to provide a percent of injected dose of 18F-fluoroclorgyline/g of wet tissue for each group of rats. Metabolite analysis was carried out in the blood plasma (75 min postinjection of 100 ␮Ci of 18 F-fluoroclorgyline). The blood samples (1 mL) were centrifuged at 1,000 ⫻ g for 4 min to separate plasma from blood cells and the plasma was removed for further analyses. The plasma (200 ␮L) was counted for total radioactivity before addition of 100 ␮L of sodium bicarbonate. The plasma mixture was then extracted with ethyl acetate (3 ⫻ 500 ␮L) and the combined ethyl acetate extracts were filtered and evaporated to dryness, and the residue was redissolved in methanol for TLC analysis. The aqueous plasma layer and combined ethyl acetate extracts were also counted in a well-counter to evaluate percent of parent remaining in the plasma. The TLC plates were developed in chloroform-methanol (9:1) and TLC analysis was performed and compared with an authentic sample of 18 F-fluoroclorgyline (Rf ⫽ 0.70 for 18F-fluoroclorgyline), and cut into 1-cm wide strips, which were then counted in a well counter. Similarly, the excised brain regions were homogenized with 5% sodium bicarbonate (500 ␮L) and centrifuged at 1,000 ⫻ g for 10 min. The aqueous layer was removed and extracted three times with ethyl acetate (500 ␮L each time). The solutions were counted and analyzed by TLC and the Rfs compared with an authentic sample of 18F-fluoroclorgyline as described above, indicating the radioactivity to be primarily the parent radioligand. RESULTS The synthesis of the precursor, N-[3-(2⬘,4⬘-dichlorophenoxy)2-mesyloxypropyl]-N-methylpropargylamine, was carried out in moderate yields in a three-step reaction starting from 2,4-dichlorophenol and 1,3-dibromo-2-propanol (reaction scheme shown in Fig. 2). The coupling of the sodium dichlorophenoxide with 1,3dibromo-2-propanol provided the desired product in approximately 40 – 45% yields. Subsequently, this product was coupled to Nmethylpropargylamine to provide the desired alcohol, N-[3-(2⬘,4⬘dichlorophenoxy)-2-hydroxypropyl]-N-methylpropargylamine in approximately 60% yields. Treatment of the mesylate with TBAF using reported procedures provided the unlabeled fluorinated derivative, N-[3-(2⬘,4⬘-dichlorophenoxy)-2-fluoropropyl]-N-methylpropargylamine (fluoroclorgyline). For radiolabeling purposes the alcohol had to be converted to the corresponding tosylate or mesylate. Tosylation of the alcohol did not provide significant amounts of the product. It was concluded that the steric effects might be playing a role in inhibiting the tosylation reaction. Subsequently, with the less bulkier group, mesylation led to the formation of the desired product. The product was found to be quite stable for use in radiolabeling reactions. The radiosynthesis of 18F-fluoroclorgyline using a 18F-fluoride-for-mesylate nucleophilic exchange proceeded smoothly with high radiochemical yields. The mixture was purified on reverse-phase HPLC to provide high specific activities of the radiolabeled product. The desired product, 18F-fluoroclorgyline, eluted at approximately 16.2 min (Fig. 3). A significantly large radiolabeled side-product, in radiochemical yields ranging between 50% and 80% of the product peak, was observed that eluted before 18F-fluoroclorgyline, at approximately 14.5 min as shown in the HPLC chromatogram in Figure 3. This unidentified radiolabeled side-product did not bind to MAO-A. It is hypothesized that under the reaction conditions, the mesylate precursor may lend itself to a nucleophilic substitution reaction by the ␤-nitrogen to form an “aziridinium intermediate.” Nucleophilic 18 F-fluoride may thus potentially interact with the aziridinium

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FIG. 4. In vitro binding of the compounds measured by using 18 F-fluoroclorgyline in rat brain homogenates. intermediate in two ways: (a) substitution at the tertiary carbon to provide the desired product, 18F-fluoroclorgyline and (b) substitution at the secondary carbon to provide the side-product. The in vitro binding profiles of clorgyline, Ro 41-1049, and (R)-deprenyl are shown in Figure 4. Clorgyline, which is a MAO-A selective agent (6), exhibited a high affinity (IC50 ⫽ 39 nM) and Ro 41-1049 exhibited lower affinity than clorgyline (IC50 ⫽ 420 nM). As expected, (R)-deprenyl, which is a potent MAO-B inhibitor (7), showed very weak binding (IC50 ⱖ 100 ␮M). This binding is indicative of the selective labeling of MAO-A sites by

FIG. 5. In vivo binding of 18F-fluoroclorgyline in the rat brains (cerebrum and cerebellum) and blood are shown as percent of injected dose/g of wet tissue. Rats were pretreated either with saline (control) or the various drugs, clorgyline (10 mg/kg), Ro-41-1049 (10 mg/kg), and (R)-deprenyl (10 mg/kg) administered intraperitoneally 90 min before the radioligand. All rats were sacrificed 75 min after intravenous injection of the radioligand, 18F-fluoroclorgyline.

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the radioligand, 18F-fluoroclorgyline, which is similar to the reported analogs of clorgyline (13). The in vivo effects of the compounds on the binding of 18Ffluoroclorgyline in the rat brains are shown in Figure 5. Binding was seen both in the cerebrum and cerebellum, with the former showing greater binding. The binding was reduced significantly by both MAO-A inhibitors, clorgyline, and Ro 41-1049. Clorgyline reduced the binding to less than 40% of the controls, whereas R0 41-1049 reduced the binding to less than 50% of the controls. The effect of the MAO-B inhibitor, (R)-deprenyl, on the binding of 18F-fluoroclorgyline was absent, consistent with the poor affinity of (R)deprenyl for MAO-A sites labeled with 18F-fluoroclorgyline in vitro. Metabolite analysis in the plasma indicated the presence of an unidentified hydrophylic component as well as the parent, 18Ffluoroclorgyline. At 75 min postinjection of 18F-fluoroclorgyline, approximately 25% of the radioactivity was found to be unchanged radiotracer in the plasma, whereas 75% of 18F-fluoroclorgyline was the hydrophilic metabolite. Ethyl acetate extracts of homogenized brain, 75 min postinjection of 18F-fluoroclorgyline, indicated ⬎90% 18 F-fluoroclorgyline with trace amounts of lipophilic constituents.

Although the ratio of specific-to-nonspecific binding of 18Ffluoroclorgyline was found to be similar to that of 11C-clorgyline, the longer half-life of fluorine-18 provides provides the ability to prolong the in vivo experiments to study the slow binding kinetics of the irreversible inhibitors. Furthermore, the longer half-life of fluorine-18 makes in vitro binding experiments possible, thus enabling their correlation with in vivo findings. Radioactivity present in the blood as seen in Figure 5 is due to the presence of 18F-fluoroclorgyline as well as a hydrophilic metabolite. It has been suggested that the alkylated propargylamines may lead to aliphatic acids as metabolites if they follow a similar metabolic pattern as deprenyl, that is, cleavage of the propargyl moiety, followed by oxidative deamination of the alkylamines (18). Based on our findings, it is conceivable that this mechanism may be operative in the case of 18F-fluoroclorgyline to result in the hydrophilic metabolite. Because MAO inhibition is one of the mechanisms of antidepressant therapy, 18F-fluoroclorgyline may potentially be a fluorinated PET radiotracer for future studies in animal models for drug evaluations. CONCLUSION

DISCUSSION We have developed a fluorine-18 analog of the irreversible inhibitor, clorgyline, to study the biodistribution of MAO-A. Using the limited structure–activity data on clorgyline analogs, we included a fluorine at the ␤-carbon in the propyl group of clorgyline, such that a fluorine-18 analog could be readily prepared by nucleophilic substitution of an appropriate leaving group. Although placing of the fluorine at the ␤-position was expected to cause a lowering of the basicity of the propargyl nitrogen, mechanistic implications of this on the affinity were not clear, because the nitrogen may not directly interact with the binding site (16). The synthesis proceeded smoothly to provide the precursor mesylate in three steps, starting from 2,4-dichlorophenol. Initial attempts to synthesize the corresponding tosylate precursor did not procced well, probably due to steric constraints on the hydroxyl group. The radiolabeling of the mesylate to provide 18F-fluoroclorgyline proceeded in moderate yields, which was purified on reverse-phase HPLC for use in in vitro and in vivo studies. It should be noted that all studies were carried out with the racemic mixture, because the ␤-position is a chiral center. Affinities of the MAO-A inhibitors, clorgyline, and Ro 41-1049 for the radioligand, 18F-fluoroclorgyline, were found to be 39 and 420 nM, respectively. These findings are in agreement with the reported relative affinities of these two compounds (15). The affinity of (R)-deprenyl, the MAO-B inhibitor, was significantly weak (IC50 ⱖ 100 ␮M), indicative of the good selectivity of 18 F-fluoroclorgyline for MAO-A sites. Distribution studies of 18F-fluoroclorgyline revealed interesting findings, somewhat similar to previous reports (10). Binding of 18 F-fluoroclorgyline in various brain regions after intravenous administration to rats was observed. Cerebrum and cerebellum showed approximately similar amounts of binding. Preadministration of clorgyline and Ro 41-1049 to the rats reduced the amount of binding of 18F-fluoroclorgyline in the various brain regions. Ratios of control versus clorgyline treated rats were approximately 2.5 in cerebrum and cerebellum, which is similar to that reported for 11 C-clorgyline in mice (8). Inhibitory effects of Ro 41-1049 were slightly lower than those of clorgyline, which is in agreement with the observed lower binding affinity of Ro 41-1049 than clorgyline. No effect was observed in rats pretreated with (R)-deprenyl.

The radiosynthesis of 18F-fluoroclorgyline was achieved in moderate radiochemical yield using a one-step direct nucleophilic displacement reaction. Binding of 18F-fluoroclorgyline showed a more than 1,000-fold selectivity for MAO-A over that of MAO-B, observed using deprenyl and clorgyline. In the rodent brain, 18F-fluoroclorgyline accumulated in areas known to contain significant amounts of MAO-A. The binding of 18F-fluoroclorgyline to MAO-A in vivo was blocked by pretreatment with clorgyline and Ro 41-1049. These results suggests that 18F-fluoroclorgyline is a potentially useful PET radiotracer for MAO-A.

Financial support was provided by U.S. Department of Energy DEFG02-98ER62540. We like to thank Robert Mintzer and Dr. Bingzhi Shi for technical assistance. References 1. Bergstrom M., Westerberg G., Kihlberg T. and Langstrom B. (1997) Synthesis of some 11C-labeled MAO-A inhibitors and their in vivo uptake kinetics in rhesus monkey brain. Nucl. Med. Biol. 24, 381–388. 2. Bergstrom M., Westerberg G. and Langstrom B. (1997) 11C-Harmine as a tracer for monoamine oxidase A (MAO-A): In vitro and in vivo studies. Nucl. Med. Biol. 24, 287–293. 3. Fowler J. S., MacGregor R. R., Wolf A. P., Arnett C. D., Dewey S. L., Schlyer D., Christman D., Logan J., Smith M. and Sachs H. (1987) Mapping human brain monoamine oxidase A and B with 11C-labeled suicide inactivators and PET. Science 235, 481– 485. 4. Hirata M., Magata Y., Ohmono Y., Saji H., Murakami K., Takagaki T., Yamamura N., Tanaka C., Konishi J. and Yokoyama A. (1995) Evaluation of radioiodinated iodoclorgyline as a SPECT radiopharmaceutical for MAO-A in the brain. Nucl. Med. Biol. 22, 175–180. 5. Hirata M., Magata Y., Ohmono Y., Saji H., Tanaka C. and Yokoyama A. (1994) Radiofluorinated clorgyline derivative for mapping MAO-A activity in brain with PET. J. Labelled Compd. Radiopharm. 35, 234 –235. 6. Johnston J. P. (1968) Some observations upon a new inhibitor of monoamine oxidase in brain tissue. Biochem. Pharmacol. 17, 1285–1297. 7. Knoll J. and Magyar K. (1972) Some puzzling pharmacological effects of monoamine oxidase inhibitors. Adv. Biochem. Psychopharmacol. 5, 393– 408. 8. MacGregor R. R., Halldin C., Fowler J. S., Wolf A. P., Arnett C. D., Langstrom B. and Alexoff D. (1985) Selective, irreversible in vivo binding of [11C]clorgyline and [11C]-L-deprenyl in mice: Potential for measurement of functional monoamine oxidase activity in brain using positron emission tomography. Biochem. Pharmacol. 34, 3207–3210. 9. Munson P. J. and Rodbard D. (1980) LIGAND: A versatile approach

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10.

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