Appl. Radiat. hot. Vol. 41. No. 8, pp. 145-152, 1990 ht. J. Radial. Appl. Instrum. Parr A

0883-2889/90

$3.00 + 0.00

Copyright Q 1990 Pergamon Press plc

Printed in Great Britain. All rights reserved

Facile Synthesis of [’ ‘ClBuprenorphine for Positron Emission Tomographic Studies of Opioid Receptors JOHN R. LEVER,‘12,* SAMUEL M. MAZZA,’ ROBERT F. DANNALS,‘.2 HAYDEN T. RAVERT,2 ALAN A. WILSON’ and HENRY N. WAGNER JR’.2 ‘Department of Environmental Health Sciences, The Johns Hopkins University School of Hygiene and Public Health, 615 N. Wolfe St, Baltimore and 2Department of Radiology, The Johns Hopkins University School of Medicine, The Johns Hopkins Hospital, 600 N. Wolfe St, Baltimore, MD 21205, U.S.A. (Received

10 August 1989; in revisedform

11 December

1989)

We have developed a simple and rapid method for the production of buprenorphine (BPN), a potent opioid partial agonist, labelled with carbon-l 1 at the 6-methoxy position. The procedure uses a precursor synthesized in high yield (89%) from BPN in two steps and employs [“Cliodomethane as the radiolabelling reagent. [“C]BPN of 97% radiochemical purity can be prepared in high specific activity (41 GBq/pmol; 1120 mCi/pmol) in a radiochemical yield of 10% at end-of-synthesis (not decay corrected). The [“C]BPN is available for use in studies of cerebral opioid receptors by positron emission tomography within 24 min from end-of-bombardment, including radiosynthesis, purification, formulation for i.v. injection and determination of specific activity.

Introduction

tors (Sadee et al., 1982; Wamsley et al., 1982; Frost et al., 1984; Rosenbaum et al., 1984; Frost et al., 1986). PET studies show that [“CIDPN binds to p, 6 and K opioid receptors in uivo in living human brain (Frost et al., 1988; Villemagne et al., 1988; Jones et al., 1988). In contrast, the cerebral opioid sites labelled by [3H]BPN in vivo in rodents do not include the S subtype and may correspond to both the p and K populations (Sadee et al., 1982, 1983). Thus, increased insight into the regional distribution and occupancy of the multiple receptors might be gained through the comparison of various PET studies using [“CIBPN to those using [“CIDPN or [“Clcarfentanil, a p-selective radioligand (Frost et al., 1985). BPN can be labelled at the C-20 methylene position of the cyclopropylmethyl moiety (Fig. 1) using [“Clcyclopropanecarbonyl chloride as the radiolabelling reagent in a process which requires 57-90 min from end-of-bombardment (EOB). (Luthra et al., 1987; Shiue et al., 1988). PET studies with [“CIBPN prepared in this fashion revealed specific labelling of opioid receptors in thalamic and striatal tissues of baboon brain although subtype differentiation was not addressed (Shiue et al., 1988). Since the radiosynthesis time is long with respect to the half-life of “C (20.4min), the development of a more facile route to [“CIBPN is desirable. In this report, we describe a convenient method for the production of [“CIBPN labelled at the 6-methoxy

During the past several years, extensive research efforts have been directed toward the identification and synthesis of radioligands suitable for localization and quantification of opioid receptors by positron emission tomography (PET). One structural class of ligands under investigation, the 4,5epoxymorphinans, includes etorphine, diprenorphine (DPN) and buprenorphine (BPN). Etorphine (Immobilon~), an exceptionally powerful agonist, and DPN (RevivonR), an antagonist in most pharmacological assays, are used in veterinary practice (Casy and Parfitt, 1986). BPN (Temgesic”), a prototypic opioid partial agonist which manifests a bell-shaped doseresponse curve (Dum and Herz, 1981) is clinically employed as an analgesic and as an agent for maintenance pharmacotherapy of heroin addiction (Mello and Mendelson, 1980; Bickel et al., 1988; Kosten and Kleber, 1988) and cocaine abuse (Kosten et al., 1989). Etorphine, DPN and BPN display high affinities in vitro for p, 6 and K opioid receptors (Sadee et al., 1982; Villiger, 1984) and exhibit pharmacokinetic properties suitable for in vivo studies of opioid recep*All correspondence should be addressed to: John R. Lever, Ph.D., Room 2001 Hume, The Johns Hopkins University School of Hygiene and Public Health, 615 N. Wolfe St, Baltimore, MD 21205, U.S.A. [Tel. (301) 955-33501. 745

JOHNR. LEVERrt al

146

CH, Fig. 1. Chemical structure of buprenorphine (BPN). Positions suited for labelling with “C are indicated by an asterisk.

III. Tetrahydrofuran (THF) was distilled under NZ from sodium benzophenone ketyl immediately prior to use. Dimethylformamide (DMF) was distilled under reduced pressure from CaH, and then redistilled under reduced pressure from BaO prior to storage in sealed ampules under argon. Iodomethane was distilled from CaHz and stored over copper shot and molecular sieves under argon. The BPN. HCI was kindly supplied as a gift (Norwich Eaton Pharmaceuticals Inc., Norwich, N.Y.) while other chemicals and solvents were obtained commercially.

I7-Cyclopropylmethyl-4,5a-epoxy18.19-dihydro3,6-dihydro.uy-7x-[(S)-l-hydroxy1,2,2-trimethylposition (Fig. 1) which employs [“Cliodomethane for propyll-6,14-endo-ethenomorphinan; (1) radiolabelling a precursor synthesized in only two steps from BPN (Lever et ol., 1988). The in rGl$o A solution of the free base of BPN (43.5 mg. evaluation of [“CIBPN labelled at the 6-methoxy 0.100 mmol) in THF (I .O mL) containing Ccl, position should prove of interest in terms of (0.10 mL, 1.0 mmol) was added dropwise to a solumetabolic stability, kinetic behavior and radiation tion of LiAlH, in THF (1.0 M. 4.0 mL) and the dosimetry inasmuch as N-decyclopropylmethylation mixture was refluxed under N? for 72 h. The reaction is the major route of peripheral metabolism of BPN was cooled and then quenched by the addition of in human beings (Cone et al., 1984). saturated aqueous Na,SO,, The mixture was diluted with water (50mL), brought to pH 9 with aqueous NaOH (0.1 M) and extracted with CHCI, . The comMaterials and Methods bined organic extracts were dried (Na$O,), filtered and then concentrated under reduced pressure. ShortProton nuclear magnetic resonance (‘H-NMR) path column chromatography on silica gel using a spectra were obtained with a Bruker WM-300 (300.13 MHz) at The Johns Hopkins University Biogradient of hexane/ethyl acetate (50/50 to 25175, v/v) physics NMR Facility. Chemical shifts are reported containing 1% triethylamine gave 41.0 mg (97%) of trio1 1 hemihydrate as a clear glass: ‘H-NMR, d 0. I I in parts per million (6) downfield relative to internal (2H, m); 0.48 (2H, m); 0.56-0.72 (lH, m); 0.72-0.87 tetramethylsilane (6 = 0.00) in deuterochloroform. (IH, m); 0.87-l .20 (4H, m); 1.05 (9H, s); I .2X (I H, “C-NMR spectra were recorded with an IBM NR-80 d,d, J = 12.8, 8.9 Hz); 1.38-1.53 (IH, m); 1.45 (3H, s); (20 MHz). Distinguishing NMR spectral features 1.62 (lH, m); 1.83-2.01 (2H, m); 2.10-2.40 (5H, m); were in accord with those of related compounds 2.55-2.65 (IH, m); 2.85 (lH, m); 2.96 (IH, d, (Fulmor et al., 1967; Carroll et al., 1976). High J= 18.1 Hz); 2.97 (IH, d, J=6.3Hz); 4.23 (IH. d. resolution electron impact mass spectroscopy J = 1.6Hz); 6.49 (lH, d, J = 8.0 Hz); 6.69 (1H. d. (HRMS) was performed with an AEI MS-30 at J = 8.0 Hz). HRMS, exact mass (m/e) calcd. for 20 eV, 170 C at the University of Minnesota Mass C2sH,,N0, (M+-H,O): 435.2773; found: 435.2730 Spectroscopy Facility. Chemical microanalyses were (100%). Anal. calcd. for C2,H,,N0,.0.5 HzO: C. determined by Atlantic Microlab, Inc., Norcross, 72.69; H, 8.71; N, 3.03; found: C, 73.01; H. 8.75: N, Georgia. Analytical thin layer chromatography 3.01. (TLC) was conducted on plastic-backed Macherey Nagel silica gel 60 F-254 plates (250 pm) and prepar17-Cyclopropylmethyl-4,5r-epo.uy18, I9-dihydro-.?ative TLC on glass-backed Analtech silica gel 60 (t-butyl-dimethylsilyloxy)-6-hydroxy-7r-[(S)-IF-254 plates (1000 or 250 pm). Short-path column hydroxy- 1,2,2-trimethylpropyl]-6.14-endo-ethenochromatography was performed under N1 pressure morphinan; (2) using E. Merck 7729 (~230 mesh) silica gel. The A solution of trio1 1 (24.5 mg, O.O54mmol), thigh performance liquid chromatography (HPLC) butyldimethylsilyl chloride (17.0 mg, 0. I 1 mmol) and equipment consisted of a Rheodyne Model 7126 automated injector. Waters Associates Model 5 10 imidazole (9.0 mg, 0.13 mmol) in DMF (0.6 mL) was stirred at ambient temperature under N, for 6 h. The pump and Model 440 ultraviolet (u.v.) absorbance mixture was diluted with saturated aqueous NaHCO, detector (254 nm), and an EE&G Ortec radioactivity and extracted with CH+Zl,. The combined organic detector comprised of a Model 449 ratemeter, extracts were dried (K&O,), filtered and then Model 575 amplifier, Model 550 single channel concentrated under reduced pressure. Preparative analyzer and a Nal(T1) crystal. Reverse-phase TLC using hexane/ethyl acetate (80120 v/v) containanalytical (4.6 x 250 mm) and semi-preparative ing 1% triethylamine as eluent provided 28.2 mg (10 x 250 mm) HPLC columns (Econosil C-18, (92%) of 2 as a white foam: ‘H-NMR, b 0.11 (2H, m); 10 pm) as well as normal-phase analytical HPLC 0.19 (3H, s); 0.20 (3H, s); 0.48 (2H, m); 0.57-0.72 (IH, columns (4.6 x 250 mm, Econosil silica, 10 pm) were m); 0.72-0.87 (IH, m); 0.87-l .I9 (2H, m); 0.99 (9H. obtained from Alltech Applied Sciences, Deerfield,

Facile synthesis

of [“C]buprenorphine

s); 1.04 (9H, s); 1.23-1.50 (2H, m); 1.41 (3H, s); 1.68 (lH, m); 1.88-2.07 (2H, m); 2.10-2.40 (5H, m); 2.62 (lH, m); 2.87 (lH, m); 2.98 (lH, d, J = 18.2 Hz); 2.98 (lH,d,J=6.4Hz);4.19(lH,d,J= 1.5Hz);4.72(lH, brs);6.47(lH,d,J=8.0Hz);6.62(lH,d,J=8.0Hz). HRMS, exact mass (m/e) calcd. for C,,H,,NO,Si: 567.3743; found: 567.3737 (M+, 4.7%). Anal. calcd. for C,,H,,NO,Si: C, 71.91; H, 9.41; N, 2.47; found: C, 71.65; H, 9.44; N, 2.46. l7-Cyclopropylmethyl-4,5a-epoxy-l8,J9-dihydro-3methoxy-6-hydroxy-7a-[(S)-l-hydroxy-l,2,2-trimethylpropyl]-6,14-endo -ethenomorphinan; iso -BPN (3) A mixture of trio1 1 (15.0 mg, 0.033 mmol), CH,I (23 mg, 0.16 mmol) and anhydrous K,CO, (20.0 mg, 0.145 mmol) in DMF (0.4 mL) was stirred at 40°C under N, for 48 h. The mixture was diluted with saturated aqueous NaHCO, and extracted with CH,Cl,. The combined organic extracts were dried (Na,SO,), filtered and the concentrated under reduced pressure. Preparative TLC using hexane/ethyl acetate (50/50 v/v) containing 1% triethylamine as eluent provided 4.0 mg of recovered trio1 1 (27%) and 10.2 mg (66%) of 3 as a colorless oil: ‘H-NMR, 6 0.11 (2H, m); 0.48 (2H, m); 0.62 (IH, m); 0.81 (lH, m); 0.95-1.55 (total 4H, 2m); 1.04 (9H, s); 1.40 (3H, s); 1.69 (lH, d,d, J= 12.7, 2.5 Hz); 1.98 (lH, d,d,d, J = 12.7, 12.7, 4.8 Hz); 2.10-2.47 (5H, m); 2.62 (IH, m); 2.87 (IH, m); 3.00 (lH, d, J = 18.2Hz); 3.00 (lH, d, J = 6.4 Hz); 3.33 (lH, br s; D,Oexchangeable); 3.87 (3H, s); 4.24 (lH, d, J = 0.8 Hz); 4.98 (lH, br, s; DzOexchangeable); 6.56 (IH, d, J = 8.1 Hz); 6.70 (lH, d, J = 8.1 Hz). HRMS, exact mass (m/e) calcd. for C,,H,,NO,: 467.3035; found: 467.3033 (M+, 31.2%). Anal. calcd. for C,,H,,NO,: C, 74.48; H, 8.84; N, 3.00; found: C, 74.42; H, 8.89; N, 2.96. 17-Cyclopropy1methy1-4,5a-epoxy-18,19-dihydro3,6-dimethoxy- 7s-[(S)-1-hydroxy-1,2,2-trimethylpropyl]-6,14-endo-ethenomorphinan; (4) To a solution of the free base of BPN (8.0mg, 0.017 mmol) in DMF (0.25 mL) at ambient temperature was added dry NaH (10 mg, 0.42 mmol) followed by CH,I (22.8 mg, 0.161 mmol). After lOmin, the mixture was brought to 8O’C and heated for 20 min. The reaction was allowed to cool, quenched by the addition of saturated aqueous NaHCO, (1 mL) and then extracted with CH&. The combined organic extracts were dried (NaSO,), filtered and then concentrated under reduced pressure. Preparative TLC using hexane/ethyl acetate (75/25 v/v) containing 1% triethylamine as eluent afforded 7.4 mg (87%) of 4 monohydrate as a colorless oil: ‘H-NMR, 6 0.11 (2H, m); 0.48 (2H, m); 0.6220.90 (2H, m); 0.90-1.20 (IH, m); 1.03 (9H, s); 1.30 (IH, d,d, J = 13.2, 9.6 Hz); 1.36 (3H, s); 1.67 (lH, d,d, J= 12.8, 2.5 Hz); 1.76-1.87 (2H, m); 1.97 (lH, d,d,d, J = 12.8, 12.8, 5.4Hz); 2.08-2.42(5H, m); 2.60 (lH, d,d, J = 5.4, 11.7 Hz); 2.89 (lH, d,d,d, J= 13.5, 10.1, 3.7Hz); 2.98 (lH, d, J = 6.5 Hz); 2.99 (lH, d, J = 18.0 Hz); 3.55 (3H, s);

747

3.87 (3H, s); 4.43 (lH, s); 5.92 (lH, br s; D,O exchangeable); 6.55 (lH, d, J = 8.0 Hz); 6.70 (IH, d, J = 8.0 Hz). HRMS, exact mass (m/e) calcd. for C,,H,3N0,: 481.3191; found: 481.3194 (M+, 5.5%). Anal. calcd. for C,,H,,NO,. 1.0 H,O: C, 72.11; H, 9.08; N, 2.80, found: C, 72.19; H, 8.72; N, 2.89. 17-Cyclopropy1methyl-4,5a-epoxy-18,19-dihydro-3hydroxy-6-methoxy7a-[(S)-1-hydroxy-1,2,2-trimethyIpropyl]-6,14-endo-ethenomorphinan; BPN A solution of silyl ether 2 (20.1 mg, 0.035 mmol) in DMF (0.5 mL) containing a stoichiometric amount of CH,I (5.0mg, 0.035 mmol) was treated with dry, oil-free NaH (10 mg, 0.42 mmol) at ambient temperature in a thick-walled glass vial. The vessel was sealed, incubated at 80°C for 3 min and quenched with aqueous HCl(0.5 mL, 1.O N). The mixture was diluted with saturated aqueous NaHCO, and extracted with CH,CI, (4 x 2 mL). The combined organic extracts were dried (Na,SO,), filtered and concentrated under reduced pressure. Analytical reverse-phase HPLC analyses of the crude reaction mixture using aqueous ammonium formate (0.1 M): acetonitrile (40: 60 v/v; 5 mL/min) with U.V. detection (254 nm) showed peaks corresponding to trio1 1 (25%) (R,, 1.77 min; k’, 3.4) dimethylated material 4 (19%) (R,, 12.49 min; k’, 30.0), and to the mono-methylated compounds BPN and iso-BPN 3 (56%) (R,, 3.84min; k’, 8.6). BPN and iso-BPN proved inseparable under a variety of reverse-phase HPLC conditions although the effects of pH, ion-pairing agents and differing mobile phase constituents were examined. The use of normal-phase silica and reverse-phase eluents (Adam et al., 1989) also was ineffectual. However, normal phase HPLC with hexane/ethyl acetate (70/30 v/v) containing 1% triethylamine as eluent (4 mL/min) with U.V. detection (285 nm) readily differentiated BPN (R,, 5.70 min; k ‘, 10.4) from iso-BPN (R,, 3.81 min; k’, 6.6). Using these conditions, iso-BPN 3 (ca 3%) was found in the mixture. Preparative TLC using hexane/ethyl acetate (67/33 v/v) containing 1% triethylamine provided 1 (4.5 mg, 28%) and 4 (2.9 mg; 17% based on 2, 34% based on CH,I) which displayed appropriate ‘HNMR spectral characteristics. With these TLC conditions, BPN and iso-BPN (3) are not resolved, and 8.9mg of a mixture of BPN and iso-BPN (54% combined yield) in a 97:3 ratio also was isolated. The ratio was determined by the relative ‘H-NMR integration of discrete absorptions and confirms the normal-phase HPLC result. The HRMS fragmentation pattern of the synthetic sample was virtually identical to that of authentic BPN. 17-Cyclopropy1methy1-4,5a-epoxy-18,19-dihydro-3hydroxy- 6-([I-‘I]-me thoxy)- 7a-[(S)- 1 -hydroxy1,2,2-trimethylpropy1]-6,14-endo-ethenomorphinan; [“C]BPN A solution of silyl ether DMF (0.2 mL) containing

2 (2.0mg, 3.5 pmol) a limiting amount

in of

748

JOHNR. LEVERet al

[“C]CH,I (0.5 equiv, 99 at%) was treated with dry NaH (3.0 mg) at 80°C for 3 min and quenched with aqueous HCI (0.15 mL, 1.O N). Reverse-phase HPLC analyses indicated 92% conversion, based on [“C]CH,I, to a mixture of monomethylated products (80%) and the dimethylated product 4 (12%). Trio1 1 (54% based on 2) completed the mass balance. By normal-phase HPLC, the ratio of [“C]BPN to iso[“C]BPN was 99: 1. The [‘)C]BPN was isolated by preparative TLC and displayed a single resonance at 52.4 ppm, the shift assigned to the 6-OCH, carbon of BPN, in the proton-decoupled 13C-NMR. The ‘HNMR spectrum was similar to an authentic sample of BPN except the 6-O[“C]CH, signal (3.53 ppm) appeared as a doublet: ‘J(“C-‘H) = 143 Hz. I7-Cycloprop~lmethyl-4,5u-epo.~y-l8,l9-dihydro-3hydroxy-6-([“Cl-methoxy)7a-[(S)-l-hydroxy1,2,2-trimethylpropy1]-6,14-endo-ethenomorphinun, [“C]BPN [“C]C02 was produced with a biomedical cyclotron (Instrument AB Scanditronix MC-16F, Uppsala, Sweden) by the “N(p,r)“C reaction, converted to [“C]CH,OH by LiAIH, reduction, and treated with aqueous HI at reflux to generate [“C]CHjI (Dannals et al., 1985). In a typical synthesis, [“C]CH,I (ca 18.5 GBq, 0.6 Ci) carried by a stream of N, (ca 50 mL/min). was trapped in a solution of 2 (2.0 mg, 3.5 pmol) in DMF (0.2mL) cooled in a dry ice/ethanol bath. The mixture was transferred via a short (5 cm) cannula to dry, oil-free NaH (3.5 mg, 0.15 mmol) and heated at 80°C for 2 min. The reaction was quenched by the addition of aqueous HCl (0.15 mL, 1.0 N) and diluted with HPLC mobile phase (0.2 mL) consisting of water: acetonitrile (40: 60 v/v) containing ammonium formate (0.1 M). The amounts of NaH and quenching solutions were chosen to provide a solution (ca pH 6.6) suitable for semi-preparative reverse-phase HPLC purification. The HPLC eluent (7 mL/min) was monitored by U.V. (254nM) and radioactivity detection. BPN (R,, 5.86 min; k’, 3.9) was well-separated from trio1 1 (R,, 2.88 min; k’, 1.4). The radioactive material corresponding to BPN was collected, concentrated to dryness in t’acuo, reconstituted with sterile physiological saline (7 mL) and passed through a 0.22 pm sterile filter (AcrodiscH, Gelman) into a sterile, pyrogen-free multi-dose vial. Sterile aqueous NaHCO, (8.4%. 3 mL) was added to provide a final formulation of pH 7.4. An aliquot was removed, assayed for total radioactivity in a dose calibrator (Capintec CRC-5) and checked by analytical reverse-phase HPLC using water: acetonitrile (30: 70 v/v) containing ammonium formate (0.1 M) as eluent (4.0 mljmin). The radioactive product (R,, 3.11 min; k’. 5.2) corresponded chromatographically to BPN. The area of the U.V. absorbance peak of carrier BPN measured by an automated integrator was (Hewlett-Packard 3390, Palo Alto, Calif.) and compared with the area of a standard sample of BPN for

determination of specific activity. A portion of the final formulation was brought to pH 9 with aqueous NaOH and extracted with ethyl acetate (2 x I mL). Normal-phase HPLC anlaysis of the organic layer showed [“C]BPN (> 97%) and iso-[“C]BPN (< 3%).

Results and Discussion The preparation of the silylated precursor required for the radiosynthesis of [“CIBPN was accomplished in two steps from BPN with an overall yield of 89% (Scheme 1). In the first step, trio1 1 was prepared in 97% yield by treatment of BPN with LiAIH, in THF containing Ccl,. These conditions for demethylation of the 6-methoxy position of 4,5-epoxymorphinans having an electron-rich substituent on the 7r-position were first reported by Kopcho and Schaeffer (1986). Presumably, aluminium complexation of the 6- and 19-oxygen functionalities of BPN activates the 6methoxy group toward attack by an unidentified nucleophile derived from Ccl,. In the second step. trio1 1 was converted to the phenolic t-butyldimethylsilyl ether 2 (92% yield) to direct alkylation toward 6-0-methylation. The tertiary 19- and 6-hydroxy groups of 2 were expected to be adequately differentiated on the basis of steric hindrance. In structurally similar compounds, the 19-hydroxy is highly resistant to alkylation (Bentley et al., 1967). In order to be able to assess whether 3-O-methylation or 3,6-di-0-methylation would be problematic. authentic samples of these potential products were synthesized (Scheme 1). Trio1 1 was converted with K+ZO, and CH,I to the 3-0-methylated material, iso-BPN (3), in 66% yield. By ‘H-NMR, the 3-0methyl resonance (3.87 ppm) of iso-BPN was distinct from the 6-O-methyl resonance (3.53 ppm) noted for BPN. Treatment of BPN with an.excess of CH,I and NaH provided dimethylated material 4 (87%) which displayed prominent ‘H-NMR resonances at 3.87 and 3.55 ppm for the O-methyl groups. Even under these latter forcing conditions, no evidence for methylation at the C-19 hydroxy was obtained. The degree of selectivity toward 6-0-alkylation was initially examined by treatment of silyl precursor 2 with NaH in DMF containing a stoichiometric amount of CH,I for 3min at 80°C (cf. Scheme 2). This gave a mixture of BPN and iso-BPN (3) in a 97: 3 ratio (54% combined yield) as well as dimethylated material 4 (34% yield based on CH,I). Trio1 1 completed the mass balance. Evidently, desilylation of intermediate 5 and precursor 2 occurs under the reaction conditions before the consumption of CHjI through the 6-0-alkylation of 2 is complete. The small amount of iso-BPN must be derived from trio1 1 which is liberated in situ because careful HPLC analysis of precursor 2 did not show any contamination by 1. The conditions to be used in eventual radiosyntheses were then more closely modelled by the use of a limiting amount, 0.5 equiv. of [“C]CH,I. Good

749

Facile synthesis of [“Clbuprenorphine

BPN NaH

K2C03

CH,I

CH$

i

Scheme

1. Synthetic

routes

leading to the non-labelled of possible methylated

conversion, 92%, of the alkylating agent to [“CIBPN (80%) and dimethylated material (12%) was found. Again, the remainder of precursor 2 suffered desilylation to give trio1 1. Although the

precursor of [“C]BPN side products.

and to authentic

samples

[13C]BPN sample was spectroscopically pure 13C-NMR) , a small amount of iso(‘H-NMR, [‘3C]BPN (ca 1%) was detected by normal-phase HPLC analysis.

BPN

NaH CH,I

1 Scheme

4

3 2. Proposed

pathways

for 6-0-alkylation,

3-0-alkylation

and 3,6-di-0-alkylation.

750

JOHN R. LEVERer al.

‘+ZH,I -___f NaH I DMF

HCl (aq.) HPLC

Ho-:

CH,

[“CIBPN

2

Scheme 3. Radiosynthesis of [“CIBPN labelled at the 6-methoxy position Based on the product distributions observed for the two trials, the majority of dimethylated material is most likely produced by 3-0-methylation of the BPN liberated from intermediate 5 although some may also rise from 6-0-methylation of iso-BPN (cf. Scheme 2). Further, when the molar ratio of precursor 2 to alkylating agent increases to just 2: 1, dimethylation is reduced and 6-0-alkylation leading to BPN is the favored pathway. Thus, in radiosyntheses conducted under no-carrier-added conditions, preferential “C-methylation of 2 at the 6-hydroxy

\

R -

II II1

2.88 min, trio1 1

position followed by loss of the silyl blocking group should yield [“CIBPN. To test this hypothesis, [“C]CH,I was trapped in a chilled DMF solution of precursor 2, the mixture was transferred by cannula to dry NaH, heated at 8O‘C for 2 min and then quenched (Scheme 3). Under the semi-preparative reverse-phase HPLC conditions employed for purification, [“CIBPN was wellseparated from trio1 1, the major non-radiolabelled product (Fig. 2). Isolation, concentration and formulation of the appropriate fraction provided an injectable solution of [“C]BPN which was determined to be sterile and pyrogen-free. The final formulation showed a single peak which corresponded to an authentic sample of BPN during reverse-phase HPLC analysis with U.V. absorbance and radiochemical detection (Fig. 3). Since BPN and iso-BPN are inseparable by reverse phase HPLC. the final formulation

UV Absorbance -

5.86 min, BPN

3.10 min, BPN

UV Absorbance

I

I

I

Fig. 2. Reverse-phase HPLC chromatograms observed during isolatron of [“CIBPN. The top tracing (u.v. absorbance, 254nm) shows that trio1 1 is well-separated from carrier BPN which corresponds to the major radiolabelled product as shown in the lower tracing (radioactivity).

-

3.11 min, [“C]BPN

Radioactivity

I I’

1 ’

Fig. 3. Reverse-phase HPLC chromatograms for an aliquot of the final formulation of [“CIBPN. The retention profiles of the carrier ligand shown in the upper tracing (u.v. absorbance, 254 nm) and the radiolabelled ligand shown in the lower tracing (radioactivrty) matched that of authentic BPN.

Facile synthesis of [“Clbuprenorphine

was also examined by normal-phase HPLC which revealed the presence of a small amount of iso-[“CIBPN (< 3%). The synthesis of [“C]CH,I and reaction with the precursor required 7 min from EOB, while the purification, formulation and specific activity determination required an additional 17 min. At end of synthesis (EOS, not decay corrected), the average radiochemical yield for three runs was lo%, based on [“C]CH,I, while the average specific activity was 41 GBq/pmol (1120 mCi/pmol). Although this synthetic route to [“CIBPN is patterned after the synthesis employed for the preparation of the structural congener, [“CIDPN (Lever er al.. 1987), the results are somewhat different. In the [“C]DPN case, treatment of the appropriate silylated precursor with 0.5 equiv of iodomethane allowed highly selective methylation at the 6-hydroxy position. Also, during production runs of [“C]DPN, iso-[“C]DPN is not detectable in the final formulation. Since the [“CIBPN precursor 2 is identical to the [“CIDPN precursor with the exception of a t-butyl group in place of a methyl group at C-19 (cf. Fig. I), one contributing factor may be additional steric hindrance to alkylation of the 6-hydroxy position of 2 imposed by the t-butyl substituent. This would allow desilylation and subsequent 3-0-methylation of the resulting trio1 to become more competitive with 6-0-methylation in the [“C]BPN situation. On account of the substantial reduction in affinity for opioid receptors which takes place upon methylation of the phenolic hydroxyl of 4,5-epoxymorphinans (Casy and Parfitt, 1986), iso-[“CIBPN would not be expected to label opioid receptors to any significant extent in vh>o and its presence during PET studies with [“CIBPN should cause only a slight elevation in non-specific binding. Therefore, the development of this synthetic route to [“CIBPN should facilitate studies designed to investigate dose-response or receptor-subtype occupancy relationships for a variety of opioid receptor ligands in viro by PET. Acknowledgements-This research was supported in part by USPHS Grant NINDS NS-I5080 and by postdoctoral fellowships to SMM through USPHS Training Grants NC1 CA-09199 and NIEHS ES-07141. The authors thank Dr J. James Frost for helpful discussions.

References Adam M. J., Grierson J. R. and Jivan S. (1989) An improved HPLC system for the analysis and purification of organic amine radiopharmaceuticals. Appl. Radial. Isol. 40, 9 1. Bentley K. W.. Hardy D. G. and Meek B. (1967) Novel analgesics and molecular rearrangements in the morphine-thebaine group. II. Alcohols derived from 6,14ethenoand 6,14-ethano-tetrahydrothebaine. J. Am. Chem. Sot. 89, 3273. Bickel W. K., Stitzer M. L., Bigelow G. E., Liebson 1. A., Jasinski D. R. and Johnson R. E. (1988) A clinical trial of buprenorphine: comparison with methadone in the

751

detoxification of heroin addicts. Clin. Pharmncol. Ther. 43, 72.

Carroll F. I., Moreland C. G., Brine G. A. and Kepler J. A. (1976) Carbon-13 nuclear magnetic resonance spectra of morphine alkaloids. J. Org. Chem. 41, 996. Casy A. F. and Parfitt R. T. (1986) 0pioid Analgesics: Chemistry and Receptors. Plenum Press, New York. Cone E. J., Gorodetzky C. W., Yousefnejad D., Buchwald W. F. and Johnson R. E. (1984) The metabolism and excretion of buprenorphine in humans. Drug Merab. Disp. 12, 577. Dannals R. F., Ravert H. T., Frost J. J., Wilson A. A., Burns H. D. and Wagner H. N. Jr (1985) Radiosynthesis of an opiate receptor binding radiotracer: [“Clcarfentanil. hr. J. Appl. Radial. ISOI. 36, 303. Dum J. E. and Herz A. (1981) In vivo receptor binding of the opiate partial agonist, buprenorphine, correlated with its agonistic and antagonistic actions. Br. J. Pharmacol. 74, 627. Frost J. J., Dannals R. F., Duelfer T., Burns H. D., Ravert H. T., Lgngstriim B.. Balasubramanian V. and Wagner H. N. Jr (1984) In vivo studies of opiate receptors. Ann. Neural. 15, S85. Frost J. J., Wagner H. N. Jr., Dannals R. F., Ravert H. T., Links J. M., Wilson A. A., Burns H. D., Wong D. F., McPherson R. W., Rosenbaum A. E., Kuhar M. J. and Snyder S. H. (1985) Imaging opiate receptors in the human brain by positron tomography. J. Compur. Assist. Tomogr. 9, 231. Frost J. J., Smith A. C. and Wagner H. N. Jr (1986) -‘HPDiprenorphine is selective for mu opiate receptors in oivo. Life Sri. 38, 1597. Frost J. J., Mayberg H. S., Sadzot B., Dannals R. F.. Lever J. R., Links J. M., Ravert H. T., Wilson A. A. and Wagner H. N. Jr (1988) Comparison of C-l 1 diprenorphine and C-11 carfentanil binding to opiate receptors in man by PET. J. Nucl. Med. 29, 796 (Abstract). Fulmor W., Lancaster J. E., Morton G. O., Brown J. J., Howell C. F., Nora C. T. and Hardy R. A. Jr (1967) Nuclear magnetic resonance studies in the 6,14-endoethenotetrahydrothebaine series. J. Am. Chem. Sot. 89, 3322. Jones A. K. P., Luthra S. K., Maziere B., Pike V. W.. Loc’h C., Crouzel C., Syrota A. and Jones T. (1988) Regional cerebral opioid receptor studies with [“CJdiprenorphine in normal volunteers. J. Neurosci. Meth. 23, 121. Kopcho J. J. and Schaeffer J. C. (1986) Selective O-demethylation of 7-(aminomethyl)-6,14-endo-ethenotetrahydrothebaine. J. Org. Chem. 51, 1620. Kosten T. R. and Kleber H. D. (1988) Buprenorphine detoxification from opioid dependence: a pilot study. Life Sci. 42, 635. Kosten T. R., Kleber H. D. and Morgan C. (1989) Role of opioid antagonists in treating intravenous cocaine abuse. Life Sci. 44, 887. Lever J. R., Dannals R. F., Wilson A. A., Ravert H. T. and Wagner H. N. Jr (1987) Synthesis of carbon-l 1 labeled diprenorphine: a radioligand for positron emission tomographic studies of opiate receptors. Tefrahedron Lerr. 28, 4015. Lever J. R., Mazza S. M., Dannals R. F., Wilson A. A., Ravert H. T., Frost J. J. and Wagner H. N. Jr (1988) Facile synthesis of carbon-l I labelled buprenorphine for PET studies of opiate receptors. J. Nucl. Med. 29, 1325 (Abstract). Luthra S. K.. Pike V. W., Brady F., Horlock P. L., Prenant C. and Crouzel C. (1987) Preparation of [“Clbuprenorphine-a potential radioligand for the study of the opiate receptor system in vivo. Appl. Radiat. Isot. 38, 65. Mello N. K. and Mendelson J. H. (1980) Buprenorphine suppresses heroin use by heroin addicts. Science 207, 657.

752

JOHNR. LEVERet al

Rosenbaum .I. S., Holford N. H. G., Richards R. A., Aman R. A. and Sadee W. (1984) Discrimination of three types of opioid binding sites in rat brain in viuo. Mol. Pharmacol. 25, 242. Sadee W., Rosenbaum J. S. and Herz A. (1982) Buprenorphine: differential interaction with opiate receptor subtypes in Go. J. Pharmacol. Exp. Ther. 223, 157. Sadee W.. Richards M. L., Grevel J. and Rosenbaum J. S. (1983) In cico characterization of four types of opioid binding sites in rat brain. Lijk Sri. 33, 187. Shiue C.-Y., Bai L.-Q., Teng R.-R., Arnett C. D., Dewey S. L., Wolf A. P., McPherson D., Fowler J. S., Holland M. .I. and Simon E. J. (1988) A direct comparison of the brain uptake of [“Clbuprenorphine and

[“Cldiprenorphine in baboon using PET. J. Nucl. Mrd. 29, 933 (Abstract). Villemagne V., Tanada S., Frost J. J., Dannals R. F.. Lever J. R., Natarajan T. K., Wilson A. A., Ravert H. T. and Wagner H. N. Jr (1988) Comparison of C-l 1 diprenorphine and C-l 1 carfentanil binding to opiate receptors in man using a dual detector system. J. Nucl. Med. 29, 921 (Abstract). Villiger J. W. (1984) Binding of buprenorphine to opiate receptors. Regulation by guanyl nucleotides and metal ions. Neuropharmacology 23, 373. Wamsley J. K., Zarbin M. A.. Young W. S. III and Kuhar M. J. (1982) Distribution of opiate receptors in the monkey brain: an autoradiographic study. Neuroscience 7, 595.