Synthesis of 2-iodo- and 2-phenyl-[11C]melatonin: potential PET tracers for melatonin binding sites

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Appl. Radiat. Isot. Vol. 49, No. 12, pp. 1573±1579, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0969-8043(98)00005-0 0969-8043/98 $19.00 + 0.00

Synthesis of 2-Iodo- and 2-Phenyl[11C]melatonin: Potential PET Tracers for Melatonin Binding Sites JIA JUN CHEN1, BRITA FIEHN-SCHULZE1, PAUL A. BROUGH2, VICTOR SNIECKUS2 and GUÈNTER FIRNAU1* Section of Radiology and Nuclear Medicine, Hamilton Health Sciences Corporation, McMaster University Medical Centre, 1200 Main Street, West, Hamilton, Ont., Canada L8N 3ZS and 2 Department of Chemistry, University of Waterloo, Waterloo, Ont., Canada

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(Received 22 September 1997; accepted 8 December 1997) Two 11 C-labelled melatonin derivatives, 2-iodo-[11 C]melatonin (2-iodo-5-methoxy-N[11 C-acetyl]-tryptamine, an agonist) and 2-phenyl-[11 C]melatonin (2-phenyl-5-methoxy-N[11 C-acetyl]tryptamine, a putative antagonist) were synthesized from [11 C]carbon dioxide. The reaction sequence was common to both compounds and consisted of three steps: (i) carbonylation of methyl magnesium bromide with [11 C]carbon dioxide, (ii) conversion of the adduct to [11 C]acetyl chloride, (iii) acetylation of the amine precursors (2-iodo-5-methoxy-tryptamine or 2-phenyl-5-methoxy-tryptamine) with [11 C]acetyl chloride. The precursors were especially prepared. The radiochemical yield was 19% for 2-iodomelatonin and 32% for 2-phenymelatonin, based on [11 C]carbon dioxide; the speci®c activity ranged from 300 to 600 mCi/ mmol. Both labelled 2-substituted-melatonins are intended to be used as radiotracers to study melatonin binding sites in man with positron emission tomography. # 1998 Elsevier Science Ltd. All rights reserved

Introduction The neurohormone melatonin (N-acetyl-5-methoxytryptamine) is rhythmically secreted into peripheral blood from several tissues as diverse as pineal gland, Haderian gland, retina and gastrointestinal tract (Huether, 1993). Melatonin levels in the blood are considered the chemical expression of darkness and food supply. The pineal gland secretes melatonin during the night; gastrointestinal tissue secretes it during fasting. Binding sites that may decode the melatonin signal have been found in many tissues of the mammalian body; for example, suprachiasmatic nucleus of the hypothalamus (Niles et al., 1979; Morgan and Williams, 1989), cerebellum (Fauteck et al., 1994), circle of Willis (Viswanathan et al., 1990), gastrointestinal tissue (Bubenik et al., 1993). Within these and other tissues a bewildering variety of melatonin binding sites seems to exist: speci®c proteins for membrane binding and transmembrane transport and binding to nuclear hormone receptors (Carlberg and Wiesenberg, 1995). The precise role of melatonin and its binding sites

*To whom all correspondence should be addressed.

(or receptors) is far from being clear. However, it is clear that melatonin is implicated in a number of physiological and pathological states in humans, for example, seasonal a€ective disorder, sleeplessness, jet lag (Ahrendt et al., 1987, 1988), onset of puberty, disorders of gastric motility and regulation of cell proliferation. Positron emission tomography (PET) has the potential to contribute to the understanding of the action of melatonin in humans during life. Generally, PET can visualize and measure binding sites if appropriate positron emitting tracers were available. Usually, labelled antagonists are preferred for receptor studies with PET. Unlike agonists, antagonists do not di€erentiate between anity states of G-protein coupled receptors. The receptor for melatonin is coupled to the G-protein. Consequently, a labelled melatonin antagonist would label all anity states. Thus, in vivo, the labelled antagonist is expected to produce a stronger signal from receptor containing tissue than would the labelled agonist. We set out to develop PET-tracers for the study of melatonin binding sites. 2-Substituted melatonins can be both agonists and antagonists (Spadoni et al., 1993).

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In vitro, 125 I-labelled 2-iodomelatonin ([125 I]IM) has become the most widely used ligand for the identi®cation and study of melatonin binding sites and/or receptors in animal tissues (Krause and Dubokovich, 1990). Most spectacularly, Reppert et al. (1988) have identi®ed melatonin receptor sites in the human hypothalamus on the basis of [125 I]IM binding in post mortem brain slices. The suprachiasmatic nucleus of the hypothalamus is considered to be associated with the biological clock. Gitler et al. (1990) have explored the use of [125 I]IM for in-vivo studies. In rats, they have found that [125 I]IM crosses the blood±brain-barrier rapidly and binds selectively to brain tissues known to contain melatonin binding sites, for example anterior pituitary and medial basal hypothalamus. Encouraged by the success of [125 I]IM we selected IM for labelling with the positron emitter 11 C (t1/2=20 min). In addition, 2-phenyl-melatonin has recently been reported to be an antagonist at the melatonin receptor site (Spadoni et al., 1993), although Garratt et al. (1995) found agonistic properties as well. Thus, 2-phenylmelatonin was included into our labelling e€ort. The strategy chosen for 11 C-labelling was radioacetylation of the amine nitrogen of the appropriate tryptamine analogs. We consider 11 C in the amide moiety of the melatonins a metabolically safe position for the label. This notion can be supported by the ®nding that 2-iodomelatonin is metabolised rather slowly (Stankov et al., 1993). After the administration of 2-iodomelatonin to rats, the authors analysed the plasma for metabolites arising from 2-iodomelatonin. They found that after 30 min. 94% was still original 2-iodomelatonin. The tryptamine precursors for the radioacetylation reaction, 2-iodo-5-methoxytryptamine (2a) and 2-phenyl-5-methoxy-tryptamine (2b) are not commercially available, thus they were prepared.

Experimental Materials and methods All reagent chemicals were purchased from Aldrich and used without further puri®cation. The target gas was a mixture of 14 N-nitrogen and 1% 16 O-oxygen from Matheson. The sweep gas, research-grade helium, was from Canadian Liquid Air. 2-Iodomelatonin was obtained from Research Biochemicals (RBI). The solvents were obtained from BDH and were of analytical or HPLC grade. Low resolution mass spectra were obtained on a VG ZAB-E mass spectrometer with samples being introduced through a direct inlet system. Ions of the samples were created either by electron impact (EI, 70 eV) or by chemical ionization with ammonia as the reactant gas (CI + NH3). Proton magnetic resonance (1 H-NMR) spectra were recorded at room temperature on a Bruker AM-500 spectrometer at 500.13 MHz. The spectra

Fig. 1. Structure of 2-iodo-5-methoxy-tryptamine (2a).

were accumulated in 16 to 120 scans in 16 K data points. A spectral width of 5000 Hz and a pulse width of 5 ms were used. The internal standard was tetramethylsilane. Synthesis of 2-iodo-5-methoxy-tryptamine (2a) This amine (2a) was synthesized by deacetylation of 2-iodomelatonin (1) by the method of Bertholet and Hirsbrunner (1985). 2-Iodomelatonin (1) (100 mg, 280 mmol), sodium hydroxide (66 mg, 1.65 mmol) and sodium dithionite (6 mg, 36 mmol) were dissolved in 1.0 ml isobutanol in a Reacti-Vial (Pierce Co.) under nitrogen in a glove bag and then heated at 1108C with stirring for up to 4 h. The formation of 2a (Fig. 1) was monitored during the reaction with TLC (silica; isopropanol/water/ammonia, 87:9:4; product (2) Rf=0.72; starting material (1) Rf=0.85). The reaction mixture was cooled to room temperature and extracted with 1.0 ml of water. The product in the isobutanol layer was isolated by repetitive preparative HPLC [stationary phase: C18PrepNovaPak 6 mm, 100  25 mm with a guard column of 10  25 mm; mobile phase: water±methanol (60:40) with 0.1% tri¯uoroacetic acid at 6 ml/min; UV-detector at 280 nm]. The eluate containg the amine (2a) was collected. The solvent was reduced under vacuum to half its volume. This solution was made alkaline with aqueous potassium carbonate and the amine (2a) was then extracted with 30 ml dichloromethane. The dichloromethane phase was washed with water, dried over sodium sulphate and then evaporated to dryness, to give the product 2a (8 mg, 42 mmol, light yellow oil 15%). For use in the radioacetylation reaction, 2a was redissolved in acetonitrile. MS (EI): 316 [M+] (2), 299 (6), 287 (54), 286 (98), 271 (16), 243 (12), 190 (15), 189 (100), 160 (24), 30 (24) MS (CI + NH3): 317 [M++1] (100), 300 (10), 261 (10), 191 (25), 162 (50), 160 (68) 1 HNMR (CD2Cl2+1 drop D20): 8.26 (br, 1H, H1); 7.21 (d, 1H, 8.81 Hz, H7); 6.99 (d, 1H, 2.37 Hz, H4); 6.76(dd, 1H, 8.78 Hz and 2.42 Hz, H6); 3.82 (s, 3H, He); 2.9 (t, 2H, 6.4 Hz, Hb); 2.80 (t, 2H, 6.7 Hz, Ha). The analytical data support the proposed structure of 2a. Synthesis of 2-phenyl-5-methoxy-tryptamine (2b) A modi®cation of the Medelung indole synthesis was employed to synthesise 2b and 1b according to the reaction sequence in Fig. 2.

Synthesis of

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C-labelled 2-substituted melatonins for PET

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Fig. 2. Synthesis of 2-phenyl-5-methoxy-tryptamine (2b).

N(4-Methoxy-2-methyl-aniline)benzoylamide (4) The general method by Houlihan et al. (1981) was adopted. A solution of 2-methyl-4-methoxyaniline, 3 (11.4 g) and triethylamine (12.0 ml) in dichloromethane (100 ml) was cooled in an ice bath. Benzoylchloride (9.3 ml) was added dropwise. A vigorous reaction started and a solid formed over time. Additional methylene chloride (40 ml) was added. The reaction mixture was allowed to warm up to room temperature and to stand for 10 h. The reaction mixture was washed with water (20 ml). The methylene chloride layer was dried over sodium sulphate. Evaporation of methylene chloride left an o€-white solid which was recrystallized from toluene, 13.6 g, 4, 68%. 2-Phenyl-5-methoxy-indole (5) A stirred solution of 4 (2.84 g, 11.8 mmol) in dry tetrahydrofurane (30 ml) was kept under nitrogen and was cooled to ÿ258C. A solution of n-butyl lithium (2.1 g, 29.5 mmol in 10 ml tetrahydrofurane) was added dropwise so that the internal temperature was less than ÿ108C. The reaction mixture was allowed to stand at room temperature over night and then was cooled in an ice bath. Hydrochloric acid (2 M, 14 ml) was added dropwise. Ether (25 ml) and water (10 ml) were added to dissolve some solids. The aqueous phase was extracted with toluene. The combined organic phases were dried over sodium sulphate. Evaporation of the solvents gave 1.86 g, 5, light orange solid, 64%.

tography ethylacetate±hexane (1:9) to give a brownish oil which crystallized, 6, 501 mg, 89%. 2-Phenyl-5-methoxy-tryptamine (2b) Under protection of argon, lithium aluminium hydride (230 mg) was added to anhydrous ether±tetrahydrofurane (1:1). 6 was dissolved in dry tetrahydrofurane (13 ml) and this solution was added dropwise into the lithium aluminium hydride-solution over 10 min. The mixture was stirred for 10 h, then quenched by cautious addition of ammonium chloride solution. The inorganic precipitate was ®ltered o€ and the tetrahydrofurane of the ®ltrate was evaporated. The residue was dissolved in ethylacetate (100 ml) and extracted with 2 M hydrochloric acid (3  25 ml). The acidic phase was separated, neutralized with 2 M aqueous sodium hydroxide and extracted with ethylacetate (3  75 ml). The combined organic phases were washed with saturated sodium chloride solution, dried over magnesium sulphate and evaporated to give a yellow oil, 2b, 403 mg, 66%. MS (EI,70 eV): 266[M+](15), 249 (20), 192 (15) MS (CI, +NH3): 267[M++1](100), 236 (10) 1 HNMR (CDCl3): 7.92(bs, 1H, H1); 7.59(m, 2H, 7.1 Hz, H2', H6'); 7.45 (t, 2H, 7.7 Hz, H3', H5'); 7.35 (m, 1H, 5.1 Hz, H4'); 7.25 (d, 1H, 9.0 Hz, H7); 7.07 (d, 1H, 2.3 Hz, H4); 6.86 (dd, 1H, 8.7 Hz; 2.4 Hz, H6); 3.87 (s, 3H, He); 3.03 (s, 4H, Ha and Hb); 1.33 ppm (bs, 2H, Hc). This analytical data supports the proposed structure of 2b (Fig. 3).

2-Phenyl-3-nitroethyl-5-methoxy-indole (6) 2-Nitro-1-acetoethane (0.29 ml) and 5 (426 mg, 2 mmol) were dissolved in xylenes (10 ml). The mixture was degassed using a mechanical vacuum pump and it was then heated under re¯ux. After 3 h further 2-nitro-1-acetoethane (0.1 ml) was added. After 4 h the solvent was removed and the remaining black oil was puri®ed by ¯ash chroma-

Fig. 3. Structure of 2-phenyl-5-methoxy-tryptamine (2b).

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2-Phenyl-melatonin (1b) A stirred solution of 2b (500 mg, 1.9 mmol) and triethylamine (0.8 ml) in tetrahydrofurane (30 ml) was cooled in an ice bath and acetic anhydride (0.4 ml, 3.8 mmol) was added dropwise. The mixture was allowed to warm up to room temperature and stirred over night. After removal of tetrahydrofurane in vacuo the residue was taken up in ethylacetate which was then washed with a saturated solution of sodium bicarbonate, followed by a wash with saturated solution of sodium chloride. Flash chromatography (silica gel, ethylacetate±cyclohexane, 7:3) and crystallization a€orded 235 mg, 1b, cream coloured solid, 40%. MS (EI, 70 eV): 308[M+] (42), 249 (45), 236 (100). MS (CI, +NH3): 309[M++1] (100), 236 (10), 326 (8). 1 H-NMR (CD2Cl2): 8.18 (bs, 1H, H1), 7.45 (dd, 2H, 8.0 Hz, H2', H6'), 7.47 (t, 2H, H5', H3'), 7.39 (t, 1H, 5.1 Hz, H4'), 7.29 (d, 8.7 Hz, H7), 7.10 (d, 1H, 2.0 Hz, H4), 6.90 (dd, 1H, 7.8 and 2.4 Hz, H6), 5.56 (bs, 1H, amide NH), 3.89 (s, 3H, He), 3.57 (m, 2H, Hb), 3.09 (m, 2H, Ha), 1.70 (s, 3H, acetyl CH3). The analytical data support the proposed structure of 1b. 11

C-Acetylation apparatus

All radioactive procedures were carried out in a remotely-controlled semi-automatic apparatus (Fig. 4). The hardware of this apparatus in the hot cell (valves, heaters, power jack, Geiger-MuÈller detectors) was controlled by a Macintosh PowerBook 100 programmed in HyperCard (Claris Corp, Santa Clara, CA) in conjunction with an

interface (Optomate Industrial I/O Subsystem, Transduction, Toronto, Ont.). The system allowed automatic programme cycles or, alternatively, stepby-step manual operation by clicking on the appropriate icon on the computer screen. Production of [11 C]CO2 No-carrier added [11 C]carbon dioxide was produced by the nuclear reaction 14 N(p, a)11 C using 10.5-MeV protons from the CTI Radioisotope Delivery System RDS-112. An aluminium-body gas target was ®lled with 210 psi of the target gas. At the end of bombardment (EOB) the target gas (14 N2 and [11 C]CO2) was slowly released from the target and, aided by a helium stream, trasnsported in to the 11 C-acetylation apparatus (Fig. 4). The gas was passed through a loop (140 cm total length of 1/16 inch o.d. stainless steel tubing) at liquid nitrogen temperature in which [11 C]CO2 remained. Typically, 150 mCi [11 C]CO2 were obtained at 20 mA for 8 min. Synthesis of 2-iodo- or 2-phenyl-[11 C]melatonin The [11 C]CO2 previously trapped in the loop, was transferred into reaction vessel 1 by warming the loop with hot air. Vessel 1 contained methylmagnesium bromide (300 mmol) in dibutyl ether (1 ml). During the transfer a stream of helium at 5 ml/min was passed through loop and vessel 1. After the 2min transfer a mixture of phthaloyldichloride (300 ml, 2.1 mmol) and 2,6-di-tert-butylpyridine (100 ml, 0.42 mmol) was added to vessel 1, followed by diethyl ether (100 ml). Vessel 1 was heated at

Fig. 4. Apparatus for remote control of

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C-acetylations.

Synthesis of

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C-labelled 2-substituted melatonins for PET

Fig. 5. Reaction sequence of

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C-acetylation.

1258C, vessels 1 and 2 were connected and the helium stream was increased to 15 ml/min. [11 C]Acetyl chloride was distilled into vessel 2, which contained the amine (2a or 2b) (1.5 mg, 4.7 mmol) in acetonitrile (1.0 ml) at room temperature. The distillation was stopped after 7 min and the content of vessel 2 was passed through two solid phase extraction cartridge (Silica SepPak, Waters) by the pressure of the helium stream, followed by a wash of ethyl ether (5 ml). Then, the 11 C-acetylated material was eluted from the cartridges with 5 ml of ethylacetate±ethanol (90:10). An aliquot of 50 ml was used for HPLC analysis. For use in biological studies, the solvent was evaporated by a stream of nitrogen upon heating. The remainder was redissolved in sterile saline with 0.5% of Tween-80 and ®ltered through a 0.22-mm sterile ®lter for injection. The radiochemical yield for 2-iodo-[11 C]melatonin was 19 23% (n = 6) and that of 2-phenyl-[11 C]melatonin was 32 2 4% (n = 9). The synthesis took 35 min from EOB (Fig. 5).

2 ml/min. The eluate was monitored by UV absorption at 280 nm and by a ¯ow-through gamma detector. The capacity factor k' of 2-phenyl[11 C]melatonin was found to be 5.30 which is identical to that of authentic 2-phenyl-melatonin. 2-Iodo[11 C]melatonin and authentic 2-iodo-melatonin coeluted at a k' of 5.96. No other 11 C compounds and UV-absorbing compounds were detected other than the solvent peak. For radio-TLC 5 ml of the eluate from the Silica Sep Paks were spotted on Silica Gel 60 F254 plates (0.2 mm thick layer). After development with ethylacetate/ethanol (97.5:2.5) the radioactive components were visualised and quantitated using System 200 Imaging Scanner from BioScan, Washington. Only one radioactive spot was detected. No UV absorbing compounds were detected when the plates were illuminated with UVlight. Both analysis methods gave the same result. The radiochemical purity for either labelled compounds was 96 2 3% (n = 15).

Identi®cation

For the determination of the speci®c activity, a 0.5-ml aliquot of the 5-ml-Sep-Pak elution (containing approximately 3 mCi 11 C) was chromatographed under the conditions described in Section 2.13. The response of the UV-detector was calibrated before with known amounts of authentic 2phenyl -and 2-iodo-melatonin. Thus, the peak area (UV detection) due to the 11 C sample was converted into the amount material in the sample. The eluted peak due to the 11 C-product was collected and its 11 C-content was measured in a dose calibrator. The speci®c activity was calculated as of end of synthesis. It ranged from 300 to 600 mCi/mmol for either 11 C-compound.

11

For mass spectral analysis of the C-products, the total eluate from the Silica Sep Paks was left for several hours to allow 11 C to decay; then the solvent was evaporated and redissolved in methanol. The mass spectra of 2-iodo-[11 C]melatonin showed the following mass fragmentation pattern: MS (CI + NH3) 359 [M++1] (54), 299 (14), 286 (17), 233 (65), 232[M+-iodine + 1] (95), 231[M+iodine] (62), 160 (82), which is consistent with that of authentic 2-iodo-melatonin. The mass spectra of 2-phenyl-[11 C]melatonin showed the following mass fragmentation pattern: MS (CI + NH3) 326 [M++NH4] (7), 309 [M++1] (100), 236 [M+CH2NHCOCH3] (10), which is consistent with that of authentic 2-phenyl-melatonin. Radiochemical Purity Radiochemical purity of the 11 C-products was assessed by both radio-HPLC and radio-TLC analysis. For HPLC a 50-ml aliquot of the Sep Pak elutions was chromatographed on a Whatman Silica column, 10 mm  50 cm, 5 mm particle size with ethylacetate:ethanol (95:5) at a ¯ow rate of

Speci®c activity

Discussion The principle of 11 C-acetylation by LeBars et al. (1987) has been modi®ed in several ways and applied to our work. First, the use of dibutyl ether (b.p. 1428C) as a solvent for the Grignard reaction and the subsequent quenching in vessel 1 allowed us to increase the distillation temperature to 1308C. This increased

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Speci®c activity (mCi/mmol) at EOS

Refs.

new tracers will facilitate the investigation of melatonin binding sites with PET.

0.235 77 43 300±600 300±600

Chirakal et al. (1988) LeBars et al. (1991) LeBars et al. (1987) present work present work

AcknowledgementsÐWe thank Dr Len Niles, McMaster University, for many discussions on the subject of melatonin. We gratefully acknowledge the ®nancial support of the Natural Sciences and Engineering Research Council of Canada, NSERC and the McArthur Foundation.

Table 1. Speci®c activities of melatonin PET tracers

Compound 4- and 6-[18 F]Fluoro-melatonin [11 C]Melatonin 6-Fluoro-[11 C]melatonin 2-Iodo-[11 C]melatonin 2-Phenyl-[11 C]melatonin

the eciency of the distillation of [11 C]acetyl chloride from vessel 1 to vessel 2. Mass spectral analyses of the solution containing the 11 C-products showed that evidence of dibutyl ether (MS (CI + NH3) m/ z = 131 [M++1]) only appeared at distillation temperatures above 1358C. Second, we found that an excess of phthaloyl dichloride (phthaloyl dichloride to methylmagnesium bromide = 7) was necessary to produce a maximum amount of [11 C]acetyl chloride. Third, acetonitrile was preferred over dichloromethane as solvent for the 11 C-amide formation in vessel 2. The more polar acetonitrile promotes the reaction by accommodating better the charged transition state and the elimination of hydrochloric acid. Also, the use of acetonitrile allowed us to pass the reaction mixture directly over the Silica Sep Paks. Fourth, the 11 C-acetylation apparatus allowed us to control the reaction sequence remotely. As a result, the preparation time was as short as 35 min from the end of bombardment. The speci®c activity of the PET tracers described in the present work is greater than that of previously reported melatonin tracers (Table 1). LeBars et al. (1991) used the 11 C-labelled native hormone melatonin for brain studies with PET. Their images show that [11 C]melatonin could not demonstrate speci®c retention of 11 C in brain regions rich melatonin receptors. Their failure may be due to the low speci®c activity of [11 C]melatonin (77 mCi/mmol). 2-Phenyl-[11 C]melatonin described in this work may have the appropriate properties for studying melatonin binding sites with PET. First, it is an antagonist (Spadoni et al., 1993) that interacts with the receptor with its higher anity than native melatonin. Ki for 2-phenyl-melatonin is 0.0596 nM (Garratt et al., 1995) which is 10-times higher than that of native melatonin (Ki=0.59 nM). Second, its speci®c activity (300±600 mCi/mmol) is at least 5times higher than that of [11 C]melatonin (77 mCi/ mmol). The number of PET tracers for melatonin binding sites has increased over the last decade. Nine 18 F- and 11 C-labelled compounds have been synthesised (Firnau et al., 1995). The variety of binding sites for melatonin in diverse tissues (brain and gut) may justify the use of more than one tracer. The

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C-labelled 2-substituted melatonins for PET

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