N-(6-18

F-Fluorohexyl)-N-Methylpropargylamine:A Fluorine-18-Labeled Monoamine Oxidase B Inhibitor

for Potential Use in PET StudiesJogeshwar Mukherjee,1 Zhi-Ying Yang1 and Robert Lew2

FRANKLIN MCLEAN INSTITUTE, DEPARTMENTS OF 1RADIOLOGY AND 2PHARMACOLOGICAL AND PHYSIOLOGICAL SCIENCES,

UNIVERSITY OF CHICAGO, CHICAGO, IL 60637, USA

ABSTRACT. We have synthesized N-(6-18F-fluorohexyl)-N-methylpropargylamine (18F-FHMP) as apositron emission tomography (PET) radiotracer for monoamine oxidase B (MAO-B). The radiosynthesiswas carried out by a fluorine-for-bromine substitution in 30–40% radiochemical yield in specific activitiesof 1–2 Ci/mmol. Selectivity for MAO-B was demonstrated by the high affinity of (R)-deprenyl (IC50 5 6.8nM) and lower affinity of clorgyline (IC50 5 1.2 mM) for the inhibition of 18F-FHMP binding in vitro in ratbrain homogenates. In vitro autoradiographic studies in rat brain slices showed localization of 18F-FHMP inregions such as the ependyma of the lateral ventricle, dorsal raphe, area postrema, and other regions such asthe cerebellum. The specific binding observed in the autoradiograms was displaced by preincubation with(R)-deprenyl. In in vivo experiments, the uptake of 18F-FHMP in the rat brains was high (0.10–0.20%injected dose/g). The binding of 18F-FHMP in the rat brain correlated with the general distribution ofMAO-B and was displaced completely by preadministration of 10 mM (R)-deprenyl. These results suggestthat 18F-FHMP is a potential PET radiotracer for MAO-B for use in in vitro and in vivo experiments. NUCL

MED BIOL 26;1:111–116, 1999. © 1998 Elsevier Science Inc.

KEY WORDS. N-(6-18F-Fluorohexyl)-N-methylpropargylamine (18F-FHMP), Monoamine oxidase B, Fluo-rine-18

INTRODUCTION

Biogenic amines in the brain are metabolized by monoamineoxidase. Two isoforms of this enzyme, monoamine oxidase A(MAO-A) and B (MAO-B) are present in the brain and are knownto have substrates such as dopamine, norepinephrine, serotonin,and other monoamines. Due to their potential role in altering levelsof various neurotransmitters, these enzymes have been targets fordrugs in the treatment of Parkinson’s disease and depression (6, 15,16). These enzymes are therefore being investigated by severalinvestigators noninvasively using radioligands developed forpositron emission tomography (PET) and single photon emissioncomputed tomography (SPECT). Carbon-11 labeled irreversibleradiotracers such as deprenyl (for MAO-B) and clorgyline (forMAO-A) have been prepared and extensively studied in humans(4, 5, 9). Iodine-123 labeled radiotracer of the reversible inhibitor,Ro 43-0463 has also been prepared for the study of MAO-B (1).

Our interest was to develop a fluorine-18 radiotracer for the studyof MAO-B to use in the evaluation of various antidepressants fortheir affinity to the enzyme in vitro and their ability to inhibit theenzyme in vivo. The longer half-life (110 min) of fluorine-18 wouldallow both the in vitro and in vivo pharmacological evaluation of thevarious drugs over an extended period of time, compared withcarbon-11 (20 min). A fluorinated analog of deprenyl has beenpreviously reported (12). The fluorine-18-labeled radiotracer selec-tively bound to MAO-B with somewhat faster clearance than

deprenyl (13). However, the multistep radiosynthesis of this fluo-rine-18 radiotracer precluded it from routine use (13).

Based on the various reports on MAO inhibitors in the literature,we decided on the development of irreversible agents due to therelatively lesser synthetic and radiosynthetic difficulties posed bythese compounds. We used the extensive structure–activity rela-tionship (SAR) reported on alkylated N-methylpropargylamines asirreversible inhibitors for MAO-B (17, 18). These alkylated deriv-atives were developed as MAO-B inhibitors to circumvent theformation of methamphetamine, a metabolite of deprenyl. Thesix-carbon chains, n-hexyl and n-1-methylhexyl derivatives, exhib-ited high affinities for MAO-B, comparable to that of deprenyl.They also demonstrated high selectivity for MAO-B over MAO-A(18). Using information from this SAR study, we envisaged placingthe fluorine at the terminal primary carbon atom to provide afluorinated MAO-B inhibitor analog of the n-hexyl derivative. Thisanalog could be developed into a 6-18F-fluorohexyl derivative as apotential radiotracer for PET studies. We report here the radiosyn-thesis, in vitro and in vivo characterization of N-(6-18F-fluorohexyl)-N-methylpropargylamine (18F-FHMP) as a potential fluorine-18PET radiotracer for MAO-B.

MATERIALS AND METHODS

N-Methylpropargylamine and dibromohexane were purchased fromAldrich Chemical Co. and (R)-deprenyl and clorgyline were pur-chased from Research Biochemicals International. All other re-agents were of analytical grade and were used without furtherpurification. Analytical and preparative thin-layer chromatographywere performed using Baker-flex silica gel IB-F and Alltech DC-Fertigplatten SIL G-200 UV254 plates, respectively. High perfor-

Address correspondence to: Dr. Jogeshwar Mukherjee at his present address:Department of Nuclear Medicine/PET, Kettering Medical Center, 3535Southern Boulevard, Kettering, OH 45429-1298, USA.

Received 2 November 1997.Accepted 30 May 1998.

Nuclear Medicine & Biology, Vol. 26, pp. 111–116, 1999 ISSN 0969-8051/99/$–see front matterCopyright © 1998 Elsevier Science Inc. PII S0969-8051(98)00061-4

mance liquid chromatography (HPLC) was carried out on a GilsonGradient System consisting of two Gilson pumps and one UVdetector with wavelength fixed at 254 nm and a radiation flowdetector with a NaI(Tl) crystal. Solvent A of the gradient was 0.01M phosphoric acid, pH 4.7, and solvent B was acetonitrile.Semi-prep (250 3 10 mm) C18 columns from Alltech AssociatesInc. were used for reverse-phase HPLC. Proton nuclear magneticresonance (NMR) spectra were obtained on a GE NMR OMEGA500 MHz spectrometer, and electron-impact mass spectra wereobtained on a VG Instruments Inc. Model 7250 mass spectrometer.Autoradiographic studies were carried out by exposing tissue sam-ples on storage phosphor screens. The autoradiograms were read byCyclone storage phosphor system (Packard Instruments Co.).

Radiosynthesis

Using the reported general method of alkylation of propargylamines(17), alkylation of N-methylpropargylamine (69 mg, 1 mmol) with1,6-dibromohexane (244 mg, 1 mmol) was carried out by refluxingthe mixture in triethylamine (100 mL) for 2 h (Fig. 1). The mixturewas purified on preparative thin layer chromatography (TLC) (9:1chloroform-methanol) to provide the pure product, N-(6-bromo-hexyl)-N-methylpropargylamine in 60% yield. NMR d ppm: 4.03(d, 2H, CH2 propargyl), 3.03 (t, 1H, CH propargyl), 2.87 (s, 3H,N-CH3), 3.25 (m, 2H, C-1), 2.70 (t, 2H, C-6); Mass spectra (m/z,%) 231, 233 (M1, 5%), 82 (C5H8N1, 100%).

Aqueous 18F-fluoride (10–100 mCi in volumes ranging from 0.2to 1.2 mL) was treated with potassium carbonate (0.34 mg) andKryptofix (1.88 mg) in a Pyrex round bottom flask. The mixture wasdried by heating at 85–90°C under a stream of nitrogen. Last tracesof water were removed azeotropically with acetonitrile (1–2 mL).Into this dried Kryptofix-K18F complex, a solution (2 mg in 200 mLof anhydrous 1:1 acetonitrile-dimethylsulfoxide) of N-(6-bromo-hexyl)-N-methylpropargylamine was added, and this mixture wasrefluxed at 85°C. After 20 min, the acetonitrile in the mixture wasdried under nitrogen and the residue was taken up in methanol(1 mL). This methanolic solution was passed through a mini-column of neutral alumina (prerinsed with methanol), followed bywashing the alumina with methanol (2 3 1 mL). The methanoliceluate was evaporated to near dryness and the residue taken up in60% methanol/0.01 M phosphoric acid (,1 mL) and injected intothe HPLC. Separation was carried out on an Alltech C18 column(250 3 10 mm), using gradient solution with 0.01 M phosphoricacid and acetonitrile (95% 0.01 phosphoric acid and 5% acetoni-trile at 0 min, flow rate of 3 mL/min to 5% 0.01 phosphoric acid and60% acetonitrile at 20 min, flow rate of 3 mL/min). The retentiontime of the radiotracer N-(6-18F-fluorohexyl)-N-methylpropar-gylamine was 10.5 min, whereas that of the bromo precursor was14.5 min. The yields varied between 30–40% (decay-corrected) inseveral experiments with a radiochemical purity .95%. The spe-cific activity of HPLC-purified N-(6-18F-fluorohexyl)-N-methyl-propargylamine varied between 1–2 Ci/mmol.

In Vitro Binding Assays

Rat brains were isolated and homogenized with a Tekmar Tissum-izer (15 s at half-maximum speed) in a 100-fold (w:v) dilution of a50 mM Tris HCl buffer, pH 7.4, containing 120 mM NaCl, 5 mMKCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM NaEDTA, and 0.1 mM Naascorbate. The homogenate was centrifuged at 12,000 3 g for 15min. at 4°C. The pellet was resuspended in the same volume ofbuffer, centrifuged a second time, and resuspended in fresh buffer ata concentration of 50 mg of tissue/mL. Each assay tube contained0.10 mL of this stock solution.

In vitro binding affinities of (R)-deprenyl and clorgyline (fromResearch Biochemicals Int.) to MAO-B in rat (Sprague–Dawley)brain homogenates was carried out by incubating various concen-trations [0.01 nM to 0.1 mM; dilutions were made with the bufferdescribed above (50 mM Tris-HCl buffer)] of compounds alongwith the radioligand, 18F-FHMP (1 mCi per sample tube; specificactivity 1 Ci/mmol). Binding was initiated by addition of the tissuehomogenate, and the tubes (with a total volume of 1 mL per sampletube in the 50 mM Tris-HCl buffer) were incubated for 1 h at 37°C.The binding was terminated by filtration using a Brandel filtrationapparatus, followed by washings with cold 50 mM Tris-HCl buffer(4 3 1 mL). Nonspecific binding was determined in the presence of10 mM (R)-deprenyl. The filters were counted in a well-counter forfluorine-18 activity. The data were analyzed using Ligand (BiosoftInc.) and IC50 values for the various drugs were obtained (11). Thebinding curves were displayed using GraFit (Erithacus SoftwareLtd.).

Autoradiography

Male Sprague–Dawley rats (250–300 g) were anesthetized andkilled and the brains were removed and frozen in isopentanepreviously immersed in dry ice. Sagittal tissue sections (30 mm) ofthe brain were cut on a Riechert-Jung Cryocut 1800 cryostat andmounted on microscope slides previously cleaned in chromic acid.Tissue sections were then stored at 220°C until required.

Tissue sections were removed from storage and were equilibratedto room temperature (22–25°C) over a period of 30–60 min.Following equilibration, tissue sections (16 adjacent slices, 8 wereused for total binding and 8 for nonspecific binding) were placed in50 mM Tris HCl buffer (pH 7.4, 37°C) containing 120 mM NaCland 5 mM KCl and incubated with 18F-FHMP (specific activity 51 Ci/mmol) at a concentration of 5 mCi/cc for 60 min at 37°C.Nonspecific binding was defined as the binding remaining in thepresence of 10 mM (R)-deprenyl. Following incubation, tissuesections were briefly rinsed and washed for 2 3 2 min periods at37°C in the above buffer. After washing, tissue sections were rinsedin distilled water and dried under a cool stream of air. Tissuesections were apposed to storage phosphor screens (Packard Instru-ments Co.) and exposed for 2 h. Autoradiograms were read usingthe Cyclone storage phosphor system (Packard Instruments Co.).

FIG. 1. Radiosynthesis scheme for N-(6-18F-fluorohexyl)-N-methylpropargylamine (18F-FHMP).

112 J. Mukherjee et al.

In Vivo Studies

For in vivo studies, groups (n 5 4) of Sprague–Dawley rats (250–300g) were administered 18F-FHMP. The radioligand, 18F-FHMP, 100mCi (specific activity 1 Ci/mmol), was administered intravenously(IV) into each rat under anesthesia. The rats were subsequentlyallowed to recover and had free access to food and water. The ratswere killed at various times (30, 60, 90, and 120 min) after theradioligand injection and the various brain regions (cerebrum andcerebellum) and blood were isolated into tared vials and counted forfluorine-18 activity to provide a measurement of percent of injecteddose of 18F-FHMP/g of wet tissue for each group of rats.

For competition studies, groups (n 5 4) of Sprague–Dawley rats(250–300 g) were preadministered with (R)-deprenyl 10 mg/kg.The drug (including saline, for control rats) was administeredintraperitoneally (IP) under anesthesia, 90 min prior to injection ofthe radioligand, and the rats were allowed free access to food andwater during the interval. The radioligand, 18F-FHMP, 100 mCi(specific activity 1 Ci/mmol), was administered IV into each ratunder anesthesia. The rats were subsequently allowed to recover andhad free access to food and water. All rats were sacrificed 90 minafter the radioligand injection and the various brain regions(cerebrum and cerebellum) and blood were analyzed as describedabove.

Metabolite Analysis

Metabolite analysis in the rats was carried out in the blood plasmaat various time intervals (1, 30, and 60 min postinjection of 100mCi of 18F-FHMP). The blood samples (1 mL) were centrifuged at1000 3 g for 4 min to separate plasma from blood cells and theplasma was removed for further analyses. The plasma (200 mL) wascounted for total radioactivity before addition of 100 mL of 5%sodium bicarbonate. The plasma mixture was then extracted withethyl acetate (3 3 500 mL) and the combined ethyl acetate extractswere filtered and evaporated to dryness, and the residue wasredissolved in methanol for TLC analysis. The aqueous plasma layerand combined ethyl acetate extracts were also counted in awell-counter to evaluate percent of parent remaining in the plasma.The TLC plates were developed in chloroform-methanol (9:1) andTLC analysis was performed and compared to an authentic sampleof 18F-FHMP (Rf 5 0.55 for 18F-FHMP), cut into 1-cm wide strips,which were then counted in a well counter. The hydrophilicmetabolite remained close to the origin, and the majority of it wasnot retained on a column of neutral alumina, indicating that themetabolite was not fluoride anion. Similarly, the excised brain washomogenized with 5% sodium bicarbonate (500 mL) and centri-fuged at 1000 3 g for 10 min. The aqueous layer was removed andextracted three times with ethyl acetate (500 mL each time). Thesolutions were counted and analyzed by TLC and the Rf s comparedto an authentic sample of 18F-FHMP (Rf 5 0.55 for 18F-FHMP), asdescribed above. The radioactivity was primarily the parent radio-ligand.

RESULTSRadiosynthesis

The synthesis of the precursor, N-(6-bromohexyl)-N-methylpropar-gylamine, was carried out in yields of approximately 60% in a singlestep reaction of N-methylpropargylamine and 1,6-dibromohexane(Fig. 1). The purified product was found to be stable for use inradiolabeling reactions. The radiolabeling reaction was carried outin a 1:1 mixture of anhydrous acetonitrile-dimethylsulfoxide, due to

the low solubility of the starting material in acetonitrile at ambienttemperature. The radiosynthesis of 18F-FHMP using a fluorine-for-bromine nucleophilic exchange (K18F-Kryptofix complex) pro-ceeded smoothly in 20 min at 85°C with high radiochemical yields(30–40% decay-corrected). The mixture was purified on reverse-phase HPLC to provide high specific activities (1–2 Ci/mmol) ofthe radiolabeled product.

In Vitro Binding Studies

The in vitro competition experiments of 18F-FHMP with (R)-deprenyl and clorgyline are shown in Figure 2. As expected,(R)-deprenyl, which is a potent MAO-B inhibitor, showed a highaffinity (IC50 5 6.8 nM). Clorgyline, which is a MAO-A selectiveagent, exhibited a significantly lower affinity (IC50 5 1.2 mM).Thus, 18F-FHMP, is approximately 200 times more selective forMAO-B over MAO-A sites. This is similar to the reported alkylatedanalogs of N-methyl-propargylamines (18).

Autoradiographic Studies

Specific binding of 18F-FHMP was seen in various regions of thecerebrum and the cerebellum in all the eight brain slices used tomeasure total binding (Fig. 3 shows an example slice). In thecerebrum, maximal binding was seen in regions such as theependyma of the lateral ventricles and dorsal raphe, with somebinding in the cortical and hippocampal regions. Of note was thebinding seen in area postrema as well, a region that is known toexhibit MAO-B binding as seen by 3H-Ro 19-6327 (14). Specificbinding was also seen in regions of cerebellum, as seen in Figure 3.This specific binding of 18F-FHMP in the cerebrum and cerebellumwas completely displaced in the presence of 10 mM (R)-deprenyl.

In Vivo Binding Studies

The time course of the binding of 18F-FHMP in rats was followed upto 120 min. Significant amounts of binding (0.10–0.20% injecteddose/g of tissue) were observed in the brain. Both cerebrum and

FIG. 2. In vitro binding of the compounds measured by usingN-(6-18F-fluorohexyl)-N-methylpropargylamine (18F-FHMP)in rat brain homogenates.

Fluorine-18 Radiotracer for Monoamine Oxidase B 113

cerebellum showed significant amounts of binding. The in vivouptake and competition of the binding of 18F-FHMP in the ratbrains are shown in Figure 4. The effect of (R)-deprenyl on thebinding of 18F-FHMP was dramatic (binding was reduced to lessthan 50% in 60–90 min compared with controls), consistent withthe high affinity of (R)-deprenyl for MAO-B in vitro. Maximalspecific to nonspecific binding ratios of the radiotracer were seenbetween 90 and 120 min postinjection. The results of metabolite

analyses of 18F-FHMP are shown in Figure 5. The radiotracer ismetabolized to hydrophilic metabolites and in 60 min postinjection,less than 10% of the radioactivity in the plasma is the intactradiotracer while the remainder is the hydrophilic metabolite. Onthe other hand, the brain showed predominantly binding of theradioligand, 18F-FHMP, indicating the nonpermeability of thehydrophilic radioligand.

DISCUSSION

Based on the reported SAR studies with alkylated propargylamines,a fluorine-18-fluorinated derivative of the n-hexyl derivative hasbeen developed. It was postulated that the fluorine atom, which issix carbon atoms removed from the propargylamine moiety, wouldbe less likely to interfere with the binding of the ligand to theenzyme. The radiolabeling of 18F-FHMP proceeded in high yieldswith only one radiochemical product, which was purified onreverse-phase HPLC for use in in vitro and in vivo studies.

Affinity of (R)-deprenyl for the radioligand, 18F-FHMP, wasfound to be 6.8 nM. The affinities of deprenyl, 2-hexyl-N-methyl-propargylamine and 1-hexyl-N-methylpropargylamine have beenreported to be 52, 82, and 310 nM, measured by inhibitingphenethylamine as a substrate for rat liver mitochondrial mem-branes (17, 18). The affinity of clorgyline, an inhibitor of MAO-A,for 18F-FHMP was found to be 1.2 mM, which is significantly lowerthan that of deprenyl, suggesting the good selectivity of 18F-FHMPfor MAO-B over MAO-A sites.

Distribution studies of 18F-FHMP using in vitro autoradiographyin rat sagittal brain slices revealed interesting findings, somewhatsimilar to what has been reported previously. The radioligandlocalized in high concentrations in the ependyma of the lateralventricle and in the dorsal raphe, regions that have previously beenshown to have greater concentrations of MAO-B (8, 14). This newradioligand, 18F-FHMP, seems to localize in the dorsal raphe andarea postrema more clearly than has been the case with the previousradioligands. The interaction of this radioligand with the lipidmicroenvironment as previously suggested (18) could be one po-tential reason for a better signal-to-noise ratio, enabling the

FIG. 3. Autoradiography of sagittal rat brain slice showing binding of N-(6-18F-fluorohexyl)-N-methylpropargylamine(18F-FHMP). Regions of greater binding were the ependyma of the lateral ventricle (E/LV), the dorsal raphe (DR), and the areapostrema (AP).

FIG. 4. In vivo binding of N-(6-18F-fluorohexyl)-N-methyl-propargylamine (18F-FHMP) in the rat brains (cerebrum andcerebellum) shown as percent of injected dose per gram ofwet tissue. Rats were pretreated either with saline (control) or(R)-deprenyl administered intraperitoneally (IP) 90 min priorto the radioligand. All rats were sacrificed 90 min afterintravenous (IV) injection of the radioligand, 18F-FHMP.

114 J. Mukherjee et al.

visualization of regions such as the dorsal raphe and area postrema.Binding was also seen in various parts of the cerebellum and cortex,similar to that reported previously with reversible radioligands,3H-Ro 19-6327 and 123I-Ro 43-0463 (1, 14). The area postremaclearly exhibited binding of 18F-FHMP, a finding that has beenshown previously by 3H-Ro 19-6327 (14). It has been reported thatthere is adrenergic and dopaminergic innervation in the areapostrema (2, 7) and that these neurons may participate in cardio-

vascular control, food intake, body weight, and fluid homeostasis (3,10). Specific binding of 18F-FHMP in the autoradiograms wascompletely displaced by (R)-deprenyl.

Binding of 18F-FHMP in various brain regions after IV adminis-tration to rats was observed. Cerebrum and cerebellum showedapproximately similar amounts of binding. Preadministration of(R)-deprenyl to the rats reduced the amount of binding of 18F-FHMP to the various brain regions. Ratios of control versusdeprenyl rats increased over time, and at 90–120 min they wereapproximately 2.5 in various parts of the brain, a finding that issimilar to that reported for 11C-deprenyl in mice (9).

A significant amount of radioactivity present in the blood as seenin Figure 5 was due to the presence of a hydrophilic metabolite.Metabolite analyses of 18F-FHMP in the rats at various timesrevealed a predominant hydrophilic radiolabeled metabolite thatdid not cross into the brain. It is possible that this metabolite maybe the 18F-fluorohexanoic acid, resulting from oxidative deamina-tion of the radioligand, which needs to be confirmed. It has beensuggested that the alkylated propargylamines may lead to aliphaticacids as metabolites if they follow a similar metabolic pattern asdeprenyl, that is, cleavage of the propargyl moiety, followed byoxidative deamination of the alkylamines (17, 18). Based on ourfindings, it is conceivable that this mechanism may be operative inthe case of 18F-FHMP.

Because MAO inhibition is one of the mechanisms of antide-pressant therapy as well as a treatment for Parkinson’s disease,18F-FHMP has the potential to be a fluorinated PET radiotracer forfuture studies in animal models for drug evaluations.

CONCLUSION

The radiosynthesis of 18F-FHMP was achieved in high radiochem-ical yield using a one-step direct nucleophilic displacement reac-tion. Binding of 18F-FHMP showed a more than 200-fold selectivityfor MAO-B over that of MAO-A, observed using deprenyl andclorgyline. In the rodent brain, 18F-FHMP accumulated in areasknown to contain significant amounts of MAO-B. The binding of18F-FHMP to MAO-B in vivo was blocked by pretreatment with(R)-deprenyl. These results suggests that 18F-FHMP is a potentiallyuseful PET radiotracer for MAO-B.

Financial support was provided by U.S. Department of Energy DE-FG02-94ER61840. We thank Robert Mintzer for technical assistanceand Dr. James Lucot for helpful discussions.

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