Chemical Synthesis and Characterization of Conjugates of a Novel

peripheral inhibitor of the enzyme catechol-O-methyltransferase (COMT), which is currently under clinical evaluation for the treatment of Parkinson's ...
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Bioconjugate Chem. 2002, 13, 1112−1118

Chemical Synthesis and Characterization of Conjugates of a Novel Catechol-O-methyltransferase Inhibitor David A. Learmonth* and Ana P. Freitas Laboratory of Chemistry, Department of Research and Development, BIAL, 4745-457 S. Mamede do Coronado, Portugal. Received April 19, 2002; Revised Manuscript Received July 1, 2002

BIA 3-202, 1-(3,4-dihydroxy-5-nitrophenyl)-2-phenylethanone 3, is a novel, reversible, and tight-binding peripheral inhibitor of the enzyme catechol-O-methyltransferase (COMT), which is currently under clinical evaluation for the treatment of Parkinson’s disease as an adjunct to current L-Dopa/peripheral decarboxylase inhibitor therapy. Chemically pure, well-characterized reference standards of conjugates of 3 were required for investigation of the routes of metabolism in several animal species (including humans) and for pharmacokinetic studies. The lack of suitable literature precedents for efficient, regioselective synthesis of nitrocatechol conjugate metabolites prompted us to develop efficient and highly selective chemical preparations of O-glucuronide and O-sulfate conjugates of 3 such that multigram quantities of excellent purity can now be conveniently synthesized. It is anticipated that these procedures could be applied to the synthesis of conjugates of other COMT inhibitors, also based on the 3-nitrocatechol pharmacophore.

INTRODUCTION

Catechol-O-methyltransferase is a magnesium-dependent enzyme, widely distributed in mammalian tissues, which is now known to play a fundamental role in the inactivation of endogenous catechols (1) and detoxification of xenobiotic catechol-based structures (2), namely, by catalyzing the transfer of a methyl group from S-adenosyl-L-methionine to a hydroxyl group of the catechol (3). Potential inhibitors of COMT have aroused great interest from medicinal chemists, based on the premise that COMT inhibition could enhance the bioavailability and thus prolong the therapeutic effect of L-Dopa (3,4-dihydroxy-L-phenylalanine) in patients suffering from Parkinson’s disease (4), by preventing Omethylation to 3-O-methyldopa (3-OMD). Of the so-called second-generation COMT inhibitors, structures containing the 3-nitrocatechol motif were discovered to be highly potent. Two of these in particular, tolcapone 1 and entacapone 2 (Figure 1), were found to have inhibition constants (Ki) in the low nanomolar range and which were additionally poor substrates for the enzyme (5, 6). 1 is characterized as an equipotent inhibitor of both central and peripheral COMT, while 2 is a purely peripheral inhibitor. Both have entered into clinical practice, although due to liver toxicity concerns 1 was recently withdrawn. It is thought that peripheral COMT inhibitors may be more effective in the treatment of Parkinson’s disease, since L-Dopa undergoes its most extensive metabolic breakdown in the periphery. Due, however, to the very short in vivo half-life of 2, we were encouraged to investigate the possibility of developing a potent and long-acting inhibitor of peripheral COMT, which eventually led to the discovery of BIA 3-202, 3 (7). The metabolic profile and excretion of 1 have been reported (8), but no synthetic details of metabolites were * To whom correspondence should be addressed: Department of Research and Development, BIAL, 4745-457 S. Mamede do Coronado, Portugal. Telephone: 351-22-9866100. Fax: 351-229866192. E-mail: [email protected].

Figure 1. Chemical structures of tolcapone (1), entacapone (2), and BIA 3-202 (3).

given. Conversely, enzyme-assisted synthesis of 3-Oglucuronide conjugates of 2 has been described (9), this procedure allowing the production of only small milligram quantities of generally average purity. The lack of literature precedent on nitrocatechol metabolism in general and the synthesis of COMT inhibitor metabolites in particular prompted us to develop efficient chemical methodologies for the regioselective synthesis of highly pure, multigram quantities of conjugates of 3 as standards for identification of metabolic products. EXPERIMENTAL PROCEDURES

Chemistry. Melting points were measured in open capillary tubes on an Electrothermal model 9100 hot stage apparatus and are uncorrected. NMR spectra were recorded on a Bruker Avance DPX (400 MHz) spectrometer with solvent used as an internal standard, and data are reported in the following order: chemical shift (parts per million), multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; and br, broad), number of protons, approximate coupling constant in hertz, and assignment of a signal. IR spectra were recorded with a Bomem Hartmann & Braun MB Series FTIR spectrometer using KBr tablets. Analytical HPLC was performed on a Gilson system equipped with a model 305 pump, a model 117 UV detector, and LiChrospher 100 RP-18 EcoCART 125-3 Cartridges (Merck) in combination with acetonitrile/ water mixtures. Reverse-phase preparative HPLC was carried out on an automated Gilson system equipped with

10.1021/bc0200327 CCC: $22.00 © 2002 American Chemical Society Published on Web 08/29/2002

Conjugates of a Novel Catechol-O-methyltransferase Inhibitor

a model 321 pump, a model 155 UV-vis detector, a model 215 liquid handler, and a Waters SymmetryPrep (RP18, 7 µm) preparative column (19 mm × 150 mm) in combination with acetonitrile/water mixtures. Analytical TLC was performed on precoated silica gel plates (either Merck 60 Kieselgel F254 or Merck RP-18 F254s) and visualized with UV light. Preparative chromatography was carried out on Merck 60 Kieselgel (0.063-0.2 mm). Elemental analyses were performed on a Fisons EA 1110 CHNS instrument, and all analyses are consistent with theoretical values to within (0.4% unless otherwise indicated. Solvents and reagents were purchased from Aldrich, E. Merck, and Fluka. 1-(3-Hydroxy-4-methoxy-5-nitrophenyl)-2-phenylethanone (5). A red suspension of 3 (1.0 g, 3.66 mmol), potassium carbonate (1.77 g, 12.82 mmol), and dimethyl sulfate (1.38 g, 10.98 mmol) in dimethylformamide (DMF, 10 mL) was stirred at 80 °C for 1 h and then allowed to cool to room temperature. The inorganic material was removed by filtration, and the filter cake was washed with DMF (2 mL). The combined filtrate was poured onto water (50 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic extracts were washed with water (20 mL) and brine (20 mL), then dried over anhydrous sodium sulfate, and filtered. Evaporation (50 °C, water aspirator pressure) afforded a red oil which was chromatographed on silica gel (2:1 petroleum ether/ethyl acetate) to give the major product as an off-white solid. Recrystallization (dichloromethane/heptane) gave the title product as white crystals (0.26 g, 25%): mp 121123 °C; IR (KBr) 3361 (OH), 1681 (CdO), 1539 cm-1 (NO2); 1H NMR (DMSO-d6) δ 8.10 (d, 1H, J ) 2.1 Hz, H-6), 7.82 (d, 1H, J ) 2.1 Hz, H-2), 7.4-7.2 (m, 5H, Ph), 6.26 (br, 1H, 3-OH), 4.25 (s, 2H, CH2), 4.01 (s, 3H, 4-OCH3); 13C NMR (DMSO-d6) δ 195.3, 151.3, 145.4, 142.9, 134.0, 132.7, 129.9, 129.5, 127.9, 120.4, 118.1, 63.2, 46.0. Anal. (C15H13NO5) C, H, N. 1-(3,4-Dimethoxy-5-nitrophenyl)-2-phenylethanone (6). A stirred suspension of 3 (0.50 g, 1.83 mmol), potassium carbonate (0.89 g, 6.41 mmol), and dimethyl sulfate (0.69 g, 5.48 mmol) in acetone (10 mL) was heated at reflux for 2 h and then cooled to room temperature. The inorganic material was removed by filtration and, the filter cake was washed with acetone (3 mL). The combined filtrate was evaporated (40 °C, water aspirator pressure) to leave a yellow oil that solidified on standing. Recrystallization (96% ethanol) afforded pale yellow crystals (0.47 g, 86%): mp 81-82 °C; IR (KBr) 1697 (CdO), 1533 cm-1 (NO2); 1H NMR (DMSO-d6) δ 8.01 (d, 1H, J ) 2.1 Hz, H-6), 7.75 (d, 1H, J ) 2.1 Hz, H-2), 7.47.25 (m, 5H, Ph), 4.28 (s, 2H, CH2), 4.05 (s, 3H, 4-OCH3), 3.96 (s, 3H, 3-OCH3); 13C NMR (DMSO-d6) δ 195.3, 154.8, 147.4, 144.8, 134.3, 132.1, 129.9, 129.5, 127.8, 117.8, 115.5, 62.7, 57.2, 45.9. Anal. (C16H15NO5) C, H, N. 1-[3-Hydroxy-5-nitro(4-O-2,3,4-triacetyl-β-D-glucuronopyranosidophenyl)]-2-phenylethanone Methyl Ester (8). To a stirred suspension of 3 (1.50 g, 5.49 mmol) and 1R-bromo sugar 7 (1.76 g, 4.63 mmol) in acetonitrile (20 mL) at room temperature was added silver(I) oxide (1.53 g, 6.62 mmol) in one portion. The resulting suspension was stirred in the dark for 1 h, and then filtered through a short Celite pad. The filter cake was washed with ethyl acetate (50 mL), and the combined filtrate was washed with water (3 × 50 mL) and brine (50 mL) and then filtered through Celite again. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated (40 °C, water aspirator pressure) to leave a dark brown oil. Chromatography over silica gel (1:1 petroleum ether/ethyl acetate) afforded the major product as a pale

Bioconjugate Chem., Vol. 13, No. 5, 2002 1113

orange solid that was recrystallized (MeOH) to give beige crystals (1.44 g, 53%): mp 153-155 °C; IR (KBr) 3400 (OH), 1755 (CdO, ester), 1690 (CdO, ketone), 1544 cm-1 (NO2); 1H NMR (DMSO-d6) δ 11.1 (vbr, 1H, 3-OH), 7.97 (d, 1H, J ) 2.1 Hz, H-6), 7.73 (d, 1H, J ) 2.1 Hz, H-2), 7.4-7.2 (m, 5H, Ph), 5.54 (d, 1H, J ) 7.9 Hz, H-1′), 5.45 (t, 1H, J ) 9.6 Hz, H-3′), 5.03 (dd, 1H, J ) 7.9 and 9.6 Hz, H-2′), 5.00 (t, 1H, J ) 9.7 Hz, H-4′), 4.48 (d, 1H, J ) 9.9 Hz, H-5′), 4.38 (s, 2H, CH2), 3.60 (s, 3H, OCH3), 2.03 (s, 3H, COCH3), 1.99 (s, 3H, COCH3), 1.97 (s, 3H, COCH3); 13C NMR (DMSO-d6) δ 196.4, 170.6, 170.3, 170.0, 167.7, 152.1, 146.6, 139.8, 135.6, 134.2, 130.8, 129.4, 127.7, 120.3, 115.6, 100.8, 71.9, 71.8, 71.8, 69.8, 53.5, 45.5, 21.4, 21.3, 21.2. Anal. (C27H27NO14‚0.5H2O) C, H, N. 1-(4-O-β-D-Glucuronopyranosido-3-hydroxy-5-nitrophenyl)-2-phenylethanone (9). To a cooled (ice bath) and stirred suspension of the glucuronate 8 (1.40 g, 2.38 mmol) in methanol (30 mL) was added dropwise an aqueous 1 N sodium hydroxide solution (24 mL, 24 mmol). The resulting red mixture was stirred in the cold for 30 min and then left at room temperature for 30 min. A small amount of undissolved material was removed by filtration, and the filtrate was added to cold water containing Amberlyst 15 ion-exchange resin so that the pH reached 2. The mixture was filtered and the resin washed with methanol and water (10 mL, 1:1). The filtrate was evaporated (50 °C, water aspirator pressure) and the residue dried over phosphorus pentoxide at high vacuum to give the title product as an amorphous yellow solid (0.99 g, 93%): mp 84-88 °C; IR (KBr) 3386 (broad, OH), 1733 (CdO, acid), 1693 (CdO, ketone), 1541 cm-1 (NO2); 1H NMR (DMSO-d6) δ 7.94 (d, 1H, J ) 2.1 Hz, H-6), 7.73 (d, 1H, J ) 2.1 Hz, H-2), 7.4-7.2 (m, 5H, Ph), 5.2 (vbr, 1H, 3-OH), 5.13 (d, 1H, J ) 7.5 Hz, H-1′), 4.38 (s, 2H, CH2), 3.6 (d, 1H, J ) 9.7 Hz, H-5′), 3.5-3.15 (m, 3H, H-2′, H-3′, H-4′); 13C NMR (DMSO-d6) δ 196.3, 170.7, 152.0, 146.7, 140.7, 135.7, 133.4, 130.8, 129.3, 127.6, 120.4, 115.6, 103.8, 77.0, 76.6, 74.5, 72.2, 45.4. Anal. (C20H19NO11‚1.5H2O) C, H, N. 1-[4-Hydroxy-5-nitro(3-O-2,3,4-triacetyl-β-D-glucuronopyranosidophenyl)]-2-phenylethanone Methyl Ester (11). To a stirred solution of 3 (3.74 g, 13.7 mmol) and trichloroacetimidate 10 (6.9 g, 14.38 mmol) in dichloromethane (130 mL) at room temperature was added dropwise boron trifluoride diethyl etherate (2.91 g, 20.55 mmol). The resulting mixture was stirred for 2 h and then poured onto water (100 mL). A small amount of unreacted 3 precipitated and was removed by filtration. The organic phase was separated and washed with a cold saturated aqueous sodium bicarbonate solution (3 × 100 mL), water (100 mL), and brine (100 mL), then dried over anhydrous sodium sulfate, filtered, and evaporated (40 °C, water aspirator pressure), leaving a yellow foam. Recrystallization (ethyl acetate/petroleum ether) afforded pale yellow crystals (4.50 g, 56%): mp 181-182 °C; IR (KBr) 3437 (OH), 1752 (CdO, ester), 1678 (CdO, ketone), 1549 cm-1 (NO2); 1H NMR (DMSO-d6) δ 8.34 (d, 1H, J ) 2.1 Hz, H-6), 7.82 (d, 1H, J ) 2.1 Hz, H-2), 7.35-7.2 (m, 5H, Ph), 5.78 (d, 1H, J ) 7.5 Hz, H-1′), 5.45 (t, 1H, J ) 9.6 Hz, H-3′), 5.17 (dd, 1H, J ) 7.7 and 9.4 Hz, H-2′), 5.11 (t, 1H, J ) 9.7 Hz, H-4′), 4.70 (d, 1H, J ) 9.9 Hz, H-5′), 4.36 (d, 1H, J ) 15.9 Hz, CH2), 4.28 (d, 1H, J ) 15.9 Hz, CH2), 3.61 (s, 3H, OCH3), 1.99 (s, 3H, COCH3), 1.98 (s, 3H, COCH3), 1.96 (s, 3H, COCH3); 13C NMR (DMSO-d6) δ 195.4, 170.6, 170.4, 170.1, 168.2, 151.5, 148.2, 138.5, 136.1, 130.6, 129.4, 127.6, 124.9, 122.5, 120.5, 99.0, 72.1, 72.1, 71.6, 69.7, 53.6, 45.1, 21.5, 21.3, 21.2. Anal. (C27H27NO14‚H2O) C, H, N.

1114 Bioconjugate Chem., Vol. 13, No. 5, 2002

1-(3-O-β-D-Glucuronopyranosido-4-hydroxy-5-nitrophenyl)-2-phenylethanone (12). Hydrolysis of glucuronate 11 (1.40 g, 2.38 mmol) was carried out as described for preparation of 9 above; the product was obtained as an amorphous yellow solid (1.02 g, 96%): mp 118-122 °C; IR (KBr) 3393 (broad, OH), 1734 (CdO, acid), 1686 (CdO, ketone), 1546 cm-1 (NO2); 1H NMR (DMSO-d6) δ 11.1 (vbr, 1H, 4-OH), 8.25 (d, 1H, J ) 1.9 Hz, H-6), 7.93 (d, 1H, J ) 1.9 Hz, H-2), 7.4-7.1 (m, 5H, Ph), 5.20 (d, 1H, J ) 7.3 Hz, H-1′), 4.44 (d, 1H, J ) 16.0 Hz, CH2), 4.29 (d, 1H, J ) 16.0 Hz, CH2), 4.04 (d, 1H, J ) 9.7 Hz, H-5′), 3.5-3.3 (m, 3H, H-2′, H-3′, H-4′); 13C NMR (DMSO-d6) δ 195.8, 171.0, 147.7, 147.3, 138.6, 135.8, 130.7, 129.4, 127.6, 127.6, 120.3, 119.5, 102.5, 76.5, 76.0, 73.8, 72.3, 45.3. Anal. (C20H19NO11‚1.5H2O) C, H, N. 1,2-Dioxo-1-[3-hydroxy-5-nitro(4-O-2,3,4-triacetylβ-D-glucuronopyranosidophenyl)]-2-phenylethane Methyl Ester (14). A stirred suspension of dione 13 (3.43 g, 11.9 mmol) and 1R-bromo sugar 7 (4.73 g, 11.9 mmol) in acetonitrile (70 mL) at room temperature was treated with silver(I) oxide (2.76 g, 11.9 mmol). The resulting suspension was stirred in the dark, and workup as described for the preparation of 8 above afforded the crude product as a brown foam. Chromatography over silica gel (2:1 to 1:1 petroleum ether/ethyl acetate) gave the major product as a yellow oil that was crystallized from dichloromethane/petroleum ether to give yellowish crystals (3.91 g, 54%): mp 117-119 °C; IR (KBr) 3384 (OH), 1756 (CdO, ester), 1678 (CdO, ketone), 1546 cm-1 (NO2); 1H NMR (DMSO-d6) δ 11.39 (br, 1H, 3-OH), 7.96 (d, 2H, o-Ph), 7.81 (d, 1H, J ) 2.1 Hz, H-6), 7.8 (t, 1H, p-Ph), 7.71 (d, 1H, J ) 2.1 Hz, H-2), 7.64 (t, 2H, m-Ph), 5.64 (d, 1H, J ) 8.0 Hz, H-1′), 5.46 (t, 1H, J ) 9.7 Hz, H-3′), 5.04 (dd, 1H, J ) 7.9 and 9.7 Hz, H-2′), 5.00 (t, 1H, J ) 9.9 Hz, H-4′), 4.50 (d, 1H, J ) 10.1 Hz, H-5′), 3.60 (s, 3H, OCH3), 2.02 (s, 3H, COCH3), 1.99 (s, 3H, COCH3), 1.98 (s, 3H, COCH3); 13C NMR (DMSO-d6) δ 194.1, 192.5, 170.6, 170.3, 170.0, 167.7, 152.6, 146.8, 141.3, 136.7, 132.9, 131.0, 130.4, 129.8, 121.3, 116.8, 100.6, 72.0, 71.8, 71.8, 69.8, 53.5, 21.4, 21.3, 21.2. Anal. (C27H25NO15) C, H, N. 1,2-Dioxo-1-[4-hydroxy-5-nitro(3-O-2,3,4-triacetylβ-D-glucuronopyranosidophenyl)]-2-phenylethane Methyl Ester (15). To a stirred solution of dione 13 (0.50 g, 1.74 mmol) and trichloroacetimidate 10 (1.25 g, 2.61 mmol) in dichloromethane (20 mL) at room temperature was added dropwise boron trifluoride diethyl etherate (0.37 g, 2.61 mmol). The resulting mixture was stirred for 2 h followed by workup as described for 11 above; recrystallization of the crude product from diethyl ether afforded orange crystals (0.56 g, 53%): mp 181-183 °C; IR (KBr) 3437 (OH), 1756 (CdO, ester), 1675 (CdO, ketone), 1555 cm-1 (NO2); 1H NMR (DMSO-d6) δ 7.97 (d, 1H, J ) 2.1 Hz, H-6), 7.90 (d, 2H, o-Ph), 7.77 (t, 1H, p-Ph), 7.61 (t, 2H, m-Ph), 7.47 (d, 1H, J ) 2.1 Hz, H-2), 5.65 (d, 1H, J ) 7.9 Hz, H-1′), 5.45 (t, 1H, J ) 9.6 Hz, H-3′), 5.09 (dd, 1H, J ) 7.7 and 9.6 Hz, H-2′), 5.06 (t, 1H, J ) 9.7 Hz, H-4′), 4.62 (d, 1H, J ) 9.7 Hz, H-5′), 3.62 (s, 3H, OCH3), 2.01 (s, 3H, COCH3), 2.00 (s, 3H, COCH3), 1.99 (s, 3H, COCH3); 13C NMR (DMSO-d6) δ 196.0, 191.3, 170.5, 170.4, 170.2, 168.3, 138.6, 136.1, 133.9, 130.5, 130.4, 127.9, 118.4, 99.0, 72.1, 72.0, 71.8, 69.9, 53.6, 21.5, 21.4, 21.3. Anal. (C27H25NO15‚3H2O) C, H, N. 1-(5-Amino-3,4-dihydroxyphenyl)-2-phenylethanone (16). To a stirred, yellow suspension of 3 (5.0 g, 18.3 mmol) in methanol (100 mL) at room temperature was added 10% palladium on charcoal (0.30 g). Hydrogen gas was bubbled through the mixture for 2 h, leading to

Learmonth and Freitas

formation of a new, gray precipitate. Dimethylformamide (40 mL) was added, and the catalyst was removed by filtration through Celite. The filtrate was evaporated (50 °C, water aspirator pressure) and the residue crystallized from dichloromethane/diethyl ether to give beige crystals (4.07 g, 91%): mp 234-237 °C; IR (KBr) 3364 (OH), 3290 (NH2), 1669 (CdO, ketone), 1540 cm-1 (NO2); 1H NMR (DMSO-d6) δ 9.2 (vbr, 2H, 3-OH, 4-OH), 7.4-7.1 (m, 5H, Ph), 6.94 (d, 1H, J ) 2.1 Hz, H-2), 6.84 (d, 1H, J ) 2.1 Hz, H-6), 4.8 (vbr, 2H, NH2), 4.12 (s, 2H, CH2); 13C NMR (DMSO-d6) δ 197.2, 145.5, 138.2, 137.9, 136.9, 130.5, 129.2, 128.7, 127.3, 108.4, 106.4, 45.2. Anal. (C14H13NO3) C, H, N. 1-[5-(N-Acetylamino)-3,4-dihydroxyphenyl]-2phenylethanone (17). A stirred suspension of 16 (1.95 g, 8 mmol) in dichloromethane (20 mL) was treated with pyridine (1.90 g, 24 mmol), acetic anhydride (2.44 g, 24 mmol), and 4-(dimethylamino)pyridine (DMAP, 0.01 g). The mildly exothermic reaction subsided after 15 min, and the reaction mixture was washed with cold 2 N HCl (100 mL) and brine (50 mL), then dried over anhydrous sodium sulfate, filtered, and evaporated (40 °C, water aspirator pressure), leaving a beige solid which was recrystallized from dichloromethane/petroleum ether to give off-white crystals (2.40 g, 81%). This product (2.35 g, 6.36 mmol) was suspended in methanol (35 mL) and cooled in an ice-water bath. An aqueous 1 N sodium hydroxide solution (13 mL, 13 mmol) was added dropwise, and the resulting brown solution was stirred for 30 min and then poured onto ice and 2 N HCl (150 mL). The mixture was extracted with dichloromethane (2 × 150 mL), and the extracts were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and evaporated (40 °C, water aspirator pressure), leaving a beige solid which was recrystallized from petroleum ether/ethyl acetate to give off-white crystals (1.20 g, 66%): mp 150-153 °C; IR (KBr) 3445 (OH), 3322 (NH), 1690 (CdO, acetyl), 1640 (CdO, ketone), 1542 cm-1 (NO2); 1H NMR (CDCl3) δ 10.95 (br, 1H, 4-OH), 9.16 (br, 1H, NH), 7.55 (d, 1H, J ) 2.1 Hz, H-6), 7.49 (d, 1H, J ) 2.1 Hz, H-2), 7.4-7.1 (m, 5H, Ph), 6.36 (br, 1H, 3-OH), 4.24 (s, 2H, CH2), 1.95 (s, 3H, COCH3); 13C NMR (CDCl3) δ 198.8, 172.8, 147.8, 142.1, 134.9, 130.2, 129.3, 129.0, 127.7, 126.7, 115.0, 113.0, 45.8, 23.5. Anal. (C16H15NO4‚0.3H2O) C, H, N. 1-[5-Amino-3-hydroxy(4-O-2,3,4-triacetyl-β-D-glucuronopyranosidophenyl)]-2-phenylethanone Methyl Ester (20). A stirred semisolution of 8 (0.81 g, 1.38 mmol) in methanol (50 mL) at room temperature was treated with 10% palladium on charcoal (0.25 g), and hydrogen gas was bubbled through the mixture for 1 h. The catalyst was removed by filtration through Celite, and the filtrate was concentrated (40 °C, water aspirator pressure). Chromatography of the residue on silica gel allowed separation of two products. The faster-running component was obtained after recrystallization from dichloromethane/diisopropyl ether as beige crystals (0.5 g, 65%), subsequently identified as the title product: mp 155-157 °C; IR (KBr) 3450 (OH), 3384 (NH2), 1755 (Cd O, ester), 1673 cm-1 (CdO, ketone); 1H NMR (DMSO-d6) δ 9.63 (br, 1H, 3-OH), 7.35-7.15 (m, 5H, Ph), 6.92 (d, 1H, J ) 2.1 Hz, H-6), 6.78 (d, 1H, J ) 2.1 Hz, H-2), 5.47 (t, 1H, J ) 9.7 Hz, H-3′), 5.26 (d, 1H, J ) 7.9 Hz, H-1′), 5.12 (dd, 1H, J ) 7.9 and 9.7 Hz, H-2′), 5.02 (m, 1H, H-4′), 4.55 (d, 1H, J ) 9.9 Hz, H-5′), 4.17 (s, 2H, CH2), 3.62 (s, 3H, OCH3), 2.02 (s, 3H, COCH3), 2.00 (s, 3H, COCH3), 1.99 (s, 3H, COCH3); 13C NMR (DMSO-d6) δ 197.9, 170.5, 170.4, 170.2, 168.3, 151.0, 144.0, 136.3, 135.6, 134.3,

Conjugates of a Novel Catechol-O-methyltransferase Inhibitor

130.6, 129.3, 127.4, 108.2, 105.5, 102.1, 72.0, 71.8, 71.6, 70.0, 53.6, 45.5, 21.6, 21.4, 21.3. Anal. (C27H29NO12) C, H, N. The slower-running component obtained from the column was recrystallized from dichloromethane/petroleum ether to give white crystals (0.15 g, 19%), subsequently identified as 1-[5-amino-3-hydroxy(4-O-2,3,4triacetyl-β-D-glucuronopyranosidophenyl)]-1-hydroxy-2phenylethane methyl ester (21): mp 172-174 °C; IR (KBr) 3450 (OH), 3411 (NH2), 1754 cm-1 (CdO, ester); 1 H NMR (CDCl3) δ 7.4-7.15 (m, 5H, Ph), 6.6 (vbr, 1H, 3-OH), 6.43, 6.40 (d, 1H, J ) 2.1 Hz, H-2), 6.34, 6.32 (d, 1H, J ) 2.1 Hz, H-2), 5.3 (m, 3H, H-2′, H-3′, H-4′), 4.88 (m, 1H, H-1′), 4.69 (m, 1H, CHOH), 4.07 (m, 1H, H-5′), 3.9 (vbr, 2H, NH2), 3.78 (s, 3H, OCH3), 2.99 (dd, 1H, J ) 4.1 and 13.8 Hz, CH2), 2.90 (dd, 1H, J ) 8.9 and 13.8 Hz, CH2), 2.14 (s, 3H, COCH3), 2.11 (s, 3H, COCH3), 2.06 (s, 3H, COCH3); 13C NMR (CDCl3) δ 170.7, 170.0, 169.7, 167.1, 150.2, 150.1, 143.6, 140.7, 140.6, 138.8, 138.7, 131.4, 131.4, 130.0, 129.1, 127.2, 106.0, 105.9, 105.1, 105.0, 102.7, 75.5, 75.4, 72.7, 72.5, 72.0, 69.3, 53.8, 46.3, 21.3, 21.2, 21.0. Anal. (C27H31NO12‚1.5H2O) C, H, N. 1-(5-Amino-4-O-β-d-glucuronopyranosido-4-hydroxyphenyl)-2-phenylethanone (18). A stirred semisolution of 20 (0.34 g, 0.6 mmol) in methanol (5 mL) was treated dropwise with an aqueous 1 N sodium hydroxide solution (6 mL, 6 mmol) at room temperature. The resulting orange solution was stirred for 2 h and then cooled in an ice-water bath. Acetic acid was added until the pH reached 4, and the solvents were evaporated (60 °C, water aspirator pressure). The residue was purified by reverse-phase preparative HPLC as described above using acetonitrile/water (2:8) as the eluent. Inorganic salt-rich initial fractions were discarded. Homogeneous fractions were combined and evaporated (60 °C, water aspirator pressure) and then dried over phosphorus pentoxide at high vacuum to give the title product as a beige solid (0.2 g, 79%): mp 120-121 °C; IR (KBr) 3431 (broad, OH), 3290 (NH2), 1682 (CdO, acid), 1640 cm-1 (CdO, ketone); 1H NMR (DMSO-d6) δ 7.4-7.15 (m, 5H, Ph), 6.88 (d, 1H, J ) 2.1 Hz, H-6), 6.78 (d, 1H, J ) 2.1 Hz, H-2), 4.52 (d, 1H, J ) 8.0 Hz, H-1′), 4.19 (s, 2H, CH2), 3.64 (d, 1H, J ) 9.4 Hz, H-5′), 3.5-3.2 (m, 3H, H-2′, H-3′, H-4′); 13C NMR (DMSO-d6) δ 198.1, 173.1, 150.9, 143.6, 136.7, 136.5, 134.6, 130.6, 129.3, 127.4, 107.5, 106.6, 106.0, 76.6, 75.9, 74.4, 72.6, 45.5. Anal. (C20H21NO9‚ 1.6H2O) C, H, N. 1-[5-Amino-4-hydroxy(3-O-2,3,4-triacetyl-β-D-glucuronopyranosidophenyl)]-2-phenylethanone Methyl Ester (22). To a stirred suspension of 11 (1.10 g, 1.87 mmol) in methanol (50 mL) at room temperature was added 10% palladium on charcoal (0.28 g). Hydrogen gas was bubbled through the reaction mixture for 30 min, and then the catalyst was removed by filtration through Celite. The filtrate was concentrated (40 °C, water aspirator pressure) to leave a foam which was recrystallized from diisopropyl ether to give white crystals (1.01 g, 97%): mp 93-94 °C; IR (KBr) 3445 (broad, OH), 3290 (NH2), 1755 (CdO, ester), 1670 cm-1 (CdO, ketone); 1H NMR (DMSO-d6) δ 7.35-7.15 (m, 5H, Ph), 7.14 (d, 1H, J ) 2.1 Hz, H-6), 7.11 (d, 1H, J ) 2.1 Hz, H-2), 5.70 (d, 1H, J ) 7.9 Hz, H-1′), 5.47 (t, 1H, J ) 9.6 Hz, H-3′), 5.15 (dd, 1H, J ) 7.6 and 9.6 Hz, H-2′), 5.08 (t, 1H, J ) 9.6 Hz, H-4′), 4.71 (d, 1H, J ) 9.9 Hz, H-5′), 4.20 (d, 1H, J ) 14.9 Hz, CH2), 4.12 (d, 1H, J ) 14.9 Hz, CH2), 3.63 (s, 3H, OCH3), 2.02 (s, 3H, COCH3), 1.99 (s, 3H, COCH3), 1.95 (s, 3H, COCH3); 13C NMR (DMSO-d6) δ 197.0, 170.6, 170.4, 170.0, 168.2, 145.0, 139.9, 139.2, 136.7, 130.5,

Bioconjugate Chem., Vol. 13, No. 5, 2002 1115

129.3, 128.6, 127.4, 110.9, 107.0, 99.0, 72.5, 72.0, 71.9, 69.9, 53.6, 45.2, 21.5, 21.4, 21.2. Anal. (C27H29NO12) C, H, N. 1-(5-Amino-3-O-β-D-glucuronopyranosido3-hydroxyphenyl)-2-phenylethanone (19). A cooled (ice-water bath) and stirred solution of 22 (0.78 g, 1.39 mmol) in methanol (14 mL) at room temperature was treated dropwise with an aqueous 1 N sodium hydroxide solution (14 mL, 14 mmol). After the mixture had been stirred in the cold for 30 min, acidification, workup, and purification as described for 18 above afforded light brown crystals (0.5 g, 86%): mp 158-163 °C; IR (KBr) 3445 (broad, OH), 3290 (NH2), 1706 (CdO, acid), 1640 cm-1 (CdO, ketone); 1H NMR (DMSO-d6) δ 7.3-7.1 (m, 6H, Ph, H-2), 7.04 (d, 1H, J ) 2.1 Hz, H-6), 4.49 (d, 1H, J ) 7.2 Hz, H-1′), 4.10 (d, 1H, J ) 14.9 Hz, CH2), 4.08 (d, 1H, J ) 14.9 Hz, CH2), 3.38 (d, 1H, J ) 9.2 Hz, H-5′), 3.3-3.1 (m, 3H, H-2′, H-3′, H-4′); 13C NMR (DMSO-d6) δ 196.3, 173.8, 146.5, 145.8, 139.3, 137.2, 130.5, 129.9, 129.2, 127.2, 111.9, 110.3, 105.8, 77.0, 75.0, 74.2, 73.0, 45.1. Anal. (C20H21NO9‚1.5H2O) C, H, N. 1-(3,4-Dihydroxy-5-nitrophenyl)-2-phenylethanone, 3-O-Pyridinium Sulfate (23). A stirred solution of 3 (3.0 g, 10.99 mmol) in pyridine (50 mL) at room temperature was treated with the sulfur trioxide-pyridine complex (5.24 g, 32.96 mmol) in portions. The resulting mixture was left stirring for 4 h and then poured slowly onto ice and 2 N HCl (300 mL). After the mixture had been stirred for 30 min, the pale yellow precipitate was filtered off and washed with water (50 mL). Recrystallization (methanol) afforded pale yellow crystals (4.32 g, 91%): mp 143-146 °C; IR (KBr) 3423 (OH), 1688 (CdO), 1550 cm-1 (NO2); 1H NMR (DMSOd6) δ 8.92 (d, 2H, o-Pyr), 8.58 (t, 1H, p-Pyr), 8.31 (d, 1H, J ) 2.2 Hz, H-6), 8.25 (d, 1H, J ) 2.2 Hz, H-2), 8.05 (t, 2H, m-Pyr), 7.35-7.15 (m, 5H, Ph), 4.35 (s, 2H, CH2); 13 C NMR (DMSO-d6) δ 195.9, 149.1, 146.8, 144.4, 143.7, 139.0, 135.9, 130.8, 129.3, 128.1, 127.6, 127.3, 125.5, 121.4, 45.2. Anal. (C14H10NO8S‚C5H6N) C, H, N, S. 1-(5-Amino-3,4-dihydroxyphenyl)-2-phenylethanone, 3-O-Pyridinium Sulfate (24). A stirred semisolution of 23 (2.0 g, 4.63 mmol) in methanol (100 mL) at room temperature was treated with 5% palladium on charcoal (0.2 g). Hydrogen gas was passed through the mixture for 3 h, and then the catalyst was removed by filtration through Celite. The filtrate was concentrated (40 °C, water aspirator pressure) to leave a slightly hygroscopic brownish solid (1.78 g, 79%): mp 68-71 °C; IR (KBr) 3423 (OH), 3230 (NH2), 1671 (CdO), 1550 cm-1 (NO2); 1H NMR (DMSO-d6) δ 8.82 (d, 2H, o-Pyr), 8.34 (t, 1H, p-Pyr), 7.85 (t, 2H, m-Pyr), 7.67 (d, 1H, J ) 2.1 Hz, H-6), 7.50 (d, 1H, J ) 2.1 Hz, H-2), 7.35-7.15 (m, 5H, Ph), 4.21 (s, 2H, CH2); 13C NMR (DMSO-d6) δ 196.5, 145.6, 145.5, 144.0, 142.0, 136.3, 130.7, 129.4, 129.3, 128.5, 127.5, 127.2, 118.8, 116.3, 45.3. Anal. (C14H12NO6S‚ C5H6N‚H2O) C, H, N, S. RESULTS AND DISCUSSION

The major metabolic pathways after oral administration of 3 in several animal species were expected to give rise to three main classes of products: O-methylated catechols and several glucuronide and sulfate conjugates (this work will be published elsewhere). We recently disclosed an efficient synthesis of 3 from commercially available vanillin (7) and have developed an improved method (10) for the demethylation of the 3-O-methylated derivative 4 (the precursor of 3). The corresponding 4-Omethyl isomer 5 was obtained by regioselective monom-

1116 Bioconjugate Chem., Vol. 13, No. 5, 2002 Scheme 1. Synthesis Metabolites of 3a

of

Putative

Learmonth and Freitas O-Methylated

Table 1.

1H

NMR Chemical Shifts (δ, ppm) of 9 and 12a

proton

9

12

2 6 8

7.7, d (J ) 2.1) 7.9, d (J ) 2.1) 4.4, s

10 11 12 1′ 2′ 3′ 4′ 5′

7.2b 7.3b 7.2b 5.1, d (J ) 7.5) 3.2b 3.2b 3.4, m 3.6, d (J ) 9.7)

7.9, d (J ) 2.1) 8.2, d (J ) 2.1) 4.4, d (J ) 16.0) 4.3, d (J ) 16.0) 7.25b 7.3b 7.25b 5.2, d (J ) 7.3) 3.4b 3.35b 3.45b 4.0, d (J ) 9.7)

Reagents: (a) (CH3O)2SO2, K2CO3, DMF, 80 °C (25%); (b) (CH3O)2SO2, K2CO3, (CH3)2CO, ∆ (86%). bSee ref 10.

a All these assignments are in agreement with HMQC, HMBC, COSY, and NOESY spectra. b Overlapped signals; δ values were measured from the HMQC spectra.

ethylation of 3 using potassium carbonate and dimethyl sulfate in warm DMF. Dimethylation to give 6 proceeded without incident in acetone (Scheme 1). A variety of methods exist for the preparation of glucuronide conjugates of phenolic compounds and have been reviewed (11). Often, traditional methods are lowyielding, giving rise to byproducts and/or mixtures of Rand β-anomers. Furthermore, regiospecific glucuronidation of substituted catechols has not thus far been reported. In agreement with the 4-O-regioselectivity of methylation above, we found that Koenigs-Knorr coupling of the 1R-bromo sugar 7 with 3 using silver oxide in acetonitrile gave the protected 4-O-β-glucuronate 8 in acceptable yield (53%, Scheme 2). The 1H NMR spectrum confirmed that only the β-anomer was formed, as seen from the coupling constant (J ) 7.5 Hz) of the anomeric proton. Hydrolysis of the acetyl protecting groups was achieved using aqueous 1 N sodium hydroxide in methanol. Acidic workup (Amberlyst 15 ion-exchange resin) and evaporation of the solvents afforded the 4-O-β-glucuronide 9 in good yield (93%) and purity (HPLC, >99%). An alternative method for enzymatic glucuronidation for direct preparation of the regioisomeric 3-O-β-glucu-

ronide 10 from 3 was required. This could not be achieved using 7 with heavy metal catalysts (Ag, Cd, and Hg salts). Model studies showed, however, that acetylation of 3, rather than alkylation, led to exclusive formation of the 3-O-acylated derivative. Further encouraged by a report that the trichloroacetimidate 10 reacts with 4-nitrophenol to afford the corresponding β-conjugate in high yield (12), we were delighted to discover that reaction of 3 with 10 in the presence of excess boron trifluoride (13) afforded exclusively the protected methyl 3-O-glucuronate 11 (βanomer only) in reasonable (56%) yield (Scheme 3). The unusually large excess of boron trifluoride (1.5 equiv) was necessary as the reaction proceeded poorly with only catalytic quantities normally utilized in glucuronide synthesis by this procedure, although a similar observation with an unrelated steroidal aglycone has been reported (14). The subsequent basic hydrolysis of 11 gave the free glucuronide 12, again in good yield and excellent chemical purity (HPLC analysis). The 1H and 13C NMR resonances (Tables 1 and 2) of compounds 9 and 12 were completely and unequivocally assigned by the concerted application of homonuclear (COSY and NOESY) and 1H-detected heteronuclear one-

a

Scheme 2. Regioselective 4-O-β-D-Glucurodination of 3 and 13a

a

Reagents: (a) Ag2O, CH3CN; (b) (i) 1 N NaOH (aq), CH3OH, 0 °C, (ii) Amberlyst 15 ion-exchange resin.

Scheme 3. Regioselective 3-O-β-D-Glucurodination of 3 and 13a

a

Reagents: (a) BF3‚OEt2, CH2Cl2; (b) (i) 1 N NaOH (aq), CH3OH, 0 °C, (ii) Amberlyst 15 ion-exchange resin.

Conjugates of a Novel Catechol-O-methyltransferase Inhibitor Table 2.

13C

carbon

9

12

carbon

9

12

carbon

1 2 3 4 5 6

133.4 120.4 152.0 140.7 146.7 115.6

127.6 119.5 147.7 147.3 138.6 120.3

7 8 9 10 11 12

196.3 45.4 135.7 130.8 129.3 127.6

195.8 45.3 135.8 130.7 129.4 127.6

1′ 2′ 3′ 4′ 5′ 6′

Bioconjugate Chem., Vol. 13, No. 5, 2002 1117

NMR Chemical Shifts (δ, ppm) of 9 and 12a 9

12

103.8 102.5 74.5 73.8 76.6 76.0 72.2 72.3 77.0 76.5 170.7 171.0

a All these assignments are in agreement with HMQC and HMBC spectra.

Figure 2. Chemical structures and numbering of glucuronides 9 and 12. Scheme 4. Synthesis of Amino Metabolites of 3a

a Reagents: (a) 10% Pd/C, CH OH, H (91%); (b) (i) Ac O, 3 2 2 pyridine, DMAP, CH2Cl2, (ii) 1 N NaOH, CH3OH (66%).

bond (HMQC) and long-range (HMBC) gradient-selected correlation experiments. The HMQC sequence was first employed to determine the direct H-C correlations and to assign the proton-containing carbons. The HMBC technique was then performed to establish the long-range H-C correlations of the quaternary aromatic carbons. COSY and NOESY experiments were also performed and confirmed these assignments. The strategy for the 1H and 13C signal assignment of these compounds was as follows. For 9 and 12 (Figure 2), both H-2 and H-6 exhibited HMBC three-bond connectivities with the quaternary carbon C-4 and also with C-6 and C-2, respectively. H-2 exhibited for both compounds an HMBC two-bond correlation with C-3 and H-6 an HMBC two-bond correlation with C-5. These results allowed complete ring assignments, which were strictly required to establish the position (3- or 4-O-) of the glucuronide substituent. For compound 12, H-1′ (δ 5.2) exhibited an HMBC connectivity with C-3 (δ 147.7), confirming the glucuronide substituent on the 3-O-position. For regioisomer

9, the corresponding connectivity is also seen and there is a further HMBC correlation between the 3-hydroxyl (δ 10.8) and C-4 (δ 140.7) that confirms the glucuronide substituent at the 4-O-position. The Koenigs-Knorr and Schmidt trichloroacetimidate methods are therefore complementary in that either the 3- or 4-O-β-glucuronides of 3 may be preferentially prepared. That this chemistry can be applied to other 3-nitrocatechols can be seen from the same reactions applied to the dione 13, which gave either the 4-O-βglucuronide precursor 14 (Koenigs-Knorr) or 3-O-βglucuronide precursor 15 (Schmidt) (Schemes 2 and 3). Reduction of the nitro moiety is a common metabolic pathway for aromatic nitro compounds (and for 1), and several potential amino conjugates of 3 were envisaged. 3 was subjected to catalytic hydrogenation at room temperature and atmospheric pressure (Pd/C, H2, MeOH), undergoing rapid reduction to amino analogue 16 (Scheme 4). Exhaustive acetylation of 16 followed by careful alkaline hydrolysis of the O-acetyl residues gave N-acetyl derivative 17. Catalytic hydrogenation (Pd/C, H2, MeOH) of nitro 4-O-β- and 3-O-β-glucuronides 9 and 12, respectively, afforded the corresponding amino 4-O-β- and 3-Oβ-glucuronides 18 and 19, respectively, as rather impure (80-90%) amorphous solids. Reverse-phase chromatography failed to considerably improve the purity of each. Surprisingly, catalytic hydrogen transfer using cyclohexene, formic acid, or ammonium formate as a hydrogen donor failed to return any 18 or 19. The suspicion that the major impurity arose from concomitant reduction of the carbonyl group was confirmed on hydrogenation of 8 under the same conditions, which gave on first attempt a separable 2:1 mixture of ketone 20 and alcohol 21. Careful monitoring of the reaction and a significant reduction in the reaction time (from 6 to 1 h), however, allowed isolation of 20 in 65% yield (Scheme 5). Basic hydrolysis and acidification with acetic acid to pH 4 followed by preparative reverse-phase HPLC to remove inorganic salts gave 18 in good yield and purity (>99%, HPLC). Nitroglucuronate 11 easily underwent hydrogenation to give 22 exclusively (97%), and subsequent basic hydrolysis gave origin to amino-3-O-β-glucuronide 19 (>99% purity) under the same conditions previously mentioned (Scheme 6). Sulfate conjugation catalyzed by sulfotransferase is also a metabolic pathway for phenolic and catecholic substrates in mammals; unsurprisingly, therefore, two sulfate conjugates, of both 3 and amine 16, were detected. Synthesis of these sulfates proved to be somewhat more complicated than glucuronidation, due to the high lability of the sulfate moiety. Sulfation of 3 with the sulfur trioxide-DMF complex occurred rapidly in a DMF solution, giving a single product (as monitored by HPLC) which, on attempted isolation, reverted back to 3. The product was, however, stable in solution at room tem-

Scheme 5. Synthesis of Amino 4-O-β-D-Glucuronide Conjugate 18a

a Reagents: (a) 10% Pd/C, CH OH, H (65% for 20 and 19% for 21); (b) (i) 1 N NaOH, CH OH, (ii) AcOH (pH 4), reverse-phase 3 2 3 preparative HPLC (79%).

1118 Bioconjugate Chem., Vol. 13, No. 5, 2002 Scheme 6. Synthesis of Amino 3-O-β-D-Glucuronide Conjugate 19a

Learmonth and Freitas ACKNOWLEDGMENT

We thank Paula C. Alves for valuable technical assistance. LITERATURE CITED

a Reagents: (a) 10% Pd/C, CH OH, H (97%); (b) (i) 1 N 3 2 NaOH, CH3OH, (ii) AcOH (pH 4), reverse-phase preparative HPLC (86%).

Scheme 7. and 16a

Synthesis of 3-O-Sulfate Conjugates of 3

a Reagents: (a) SO -pyridine complex, pyridine (91%); (b) 5% 3 Pd/C, CH3OH, H2 (79%).

perature. Reaction of 3 with the pyridine-sulfur trioxide complex also gave a single product, which precipitated during the workup (Scheme 7). This was perfectly stable and could be readily isolated and recrystallized (>99% purity). 1H and 13C NMR showed that the product was exclusively the 3-O-pyridinium sulfate 23; compared to that of 3, the aromatic proton on C-2 (δ 8.25) of 23 was deshielded by the 3-sulfate ester (∆δ ) 0.55 ppm). The 13 C spectrum also exhibits a significant deshielding effect on the ortho and para carbon atoms. C-2 has a carbon shift ∆δ of 7.3 ppm, C-6 4.0 ppm, and C-4 2.5 ppm. Similar behavior has been reported with dopamine and its 3-O- and 4-O-sulfates (15). Reduction of the nitro group under catalytic hydrogenation conditions afforded the corresponding hygroscopic amino-3-O-sulfate 24 in reasonable yield (78%) and good purity (>98%). CONCLUSIONS

Efficient chemical methods suitable for the regioselective gram-scale synthesis of highly pure putative Omethyl, O-glucuronide, and O-sulfate metabolic conjugates of BIA 3-202, 1-(3,4-dihydroxy-5-nitrophenyl)-2phenylethanone 3, have been developed. These compounds are currently used as standards for the determination of the metabolic profile of 3 in various species, including humans. It is foreseen that these procedures will be useful for the synthesis of conjugates of other COMT inhibitors based on the nitrocatechol pharmacophore.

(1) Guldberg, H., and Marsden, C. (1975) Catechol-O-methyltransferase: pharmacological aspects and physiological role. Pharmacol. Rev. 27, 135-206. (2) Zhu, B. T., Ezell, E. L., and Liehr, J. G. (1994) Catechol-Omethyltransferase-catalysed rapid O-methylation of mutagenic flavanoids. Metabolic inactivation as a possible reason for their lack of carcinogenicity in vitro. J. Biol. Chem. 269, 292-299. (3) Axelrod, J., and Tomchick, R. (1958) Enzymatic O-methylation of epinephrine and other catechols. J. Biol. Chem. 233, 702-705. (4) Mannisto, P. T., and Kaakkola, S. (1990) Rationale for selective COMT inhibitors as adjuncts in the drug treatment of Parkinson’s disease. Pharmacol. Toxicol. 66, 317-323. (5) Borgulya, J., Da Prada, M., Dingemanse, J., Scherschlicht, R., Schlappi, B., and Zurcher, G. (1991) Ro 40-7592. CatecholO-methyltransferase (COMT) inhibitor. Drugs Future 16, 719-721. (6) Nissinen, E., Linde´n, I.-B., Schultz, E., and Pohto, P. (1992) Biochemical and pharmacological properties of a peripherally acting catechol-O-methyltransferase inhibitor Entacapone. Naunyn-Schmiedebergs Arch. Pharmacol. 346, 262-266. (7) Learmonth, D. A., Vieira-Coelho, M. A., Benes, J., Alves, P. C., Borges, N., Freitas, A. P., and Soares-da-Silva, P. (2002) Synthesis of 1-(3,4-dihydroxy-5-nitrophenyl)-2-phenyl-ethanone and derivatives as potent and long-acting peripheral inhibitors of catechol-O-methyltransferase. J. Med. Chem. 45 (3), 685-695. (8) Jorga, K., Fotteler, B., Heizmann, P., and Gasser, R. (1999) Metabolism and excretion of tolcapone, a novel inhibitor of catechol-O-methyltransferase. Br. J. Clin. Pharmacol. 48, 513-520. (9) Luukkanen, L., Kilpelainen, I., Kangas, H., Ottoila, P., Elovaara, E., and Taskinen, J. (1999) Enzyme-assisted synthesis and structural characterization of nitrocatechol glucuronides. Bioconjugate Chem. 10, 150-154. (10) Learmonth, D. A., and Alves, P. C. (2002) Improved method for demethylation of nitro-catechol methyl ethers. Synth. Commun. 32 (4), 641-649. (11) Stachulski, A. V., and Jenkins, G. N. (1998) The synthesis of O-glucuronides. Nat. Prod. Rep., 173-186. (12) Fischer, B., Nudelman, A., Ruse, M., Herzig, J., Gottlieb, H. E., and Keinan, E. (1984) A novel method for stereoselective glucuronidation. J. Org. Chem. 49, 4988-4993. (13) Schmidt, R. R., and Grundler, G. (1981) Simple synthesis of β-D-glucopyranosyluronates. Synthesis, 885-887. (14) Ferguson, J. R., Harding, J. R., Lumbard, K. W., Scheinmann, F., and Stachulski, A. V. (2000) Glucuronide and sulfate conjugates of ICI 182,780, a pure anti-estrogenic steroid. Order of addition, catalysis and substitution effects in glucuronidation. Tetrahedron Lett. 41, 389-392. (15) Jain, T., Kaiser, C., Ackerman, D. M., Ladd, D. L., Fong, K. L., Roberts, G. D., Staiger, D. B., Davis, L. L., and Webb, R. L. (1986) Sulfoconjugation of dopamine. A critical examination of the reaction between dopamine and concentrated sulphuric acid. Can. J. Chem. 64, 2418-2422.

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