A Concise Synthesis of the 3-O-β-d- and 4'-O-β-d ... - ACS Publications

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Bioconjugate Chem. 2003, 14, 262−267

A Concise Synthesis of the 3-O-β-D- and 4′-O-β-D-Glucuronide Conjugates of trans-Resveratrol David A. Learmonth* Laboratory of Chemistry, Department of Research & Development, BIAL, A Å Avenida da Siderurgia Nacional, 4745-457 S. Mamede do Coronado, Portugal. Received July 22, 2002; Revised Manuscript Received October 7, 2002

trans-Resveratrol ((E)-3,4′,5-trihydroxystilbene, 1) is a phytoalexin produced naturally in plants and grape skins as a stress metabolite protecting against fungal attack. Widespread interest in this apparently structurally simple molecule and synthetic stilbene analogues has arisen in recent years due to the discovery of its antioxidant, antiinflammatory, and anti-carcinogenic activities, among others. Although O-conjugation with glucuronic acid in vivo is known to represent a significant metabolic pathway for polyphenolic compounds in general and 1 in particular, preclinical studies have been hampered by the lack of chemically pure, completely characterized reference standards of both regioisomeric 3-O-β-D- and 4′-O-β-D-glucuronide conjugates of 1 for adequate identification and quantification of these significant metabolites. The present work describes a concise, convergent synthesis of both 3-O-β-D- and 4′-O-β-D-glucuronide conjugates of 1 using a strategy based on a novel Heck coupling of iodoaryl-O-β-D-glucuronate esters with appropriately substituted styrenes, such that highly pure multimilligram to gram quantities of both the 3-O-β-D- and 4′-O-β-D-glucuronide conjugates of 1 can now be conveniently synthesized.

INTRODUCTION

trans-Resveratrol ((E)-3,4′,5-trihydroxystilbene, 1) (Figure 1) is an intriguing polyhydroxy-stilbene derivative of apparent structural simplicity that has been detected in several plants (1), peanuts (2), and most abundantly in the skins of red grapes (3), where it is produced as a phytoalexin (plant antibiotic) as part of the defense mechanism against attack by the fungal grapevine pathogen Bortrytis cinerea (4). Widespread interest concerning resveratrol is manifested by an increasing number of reports describing its broad range of biological activities. Indeed, initial motivation behind research into 1 began with the discovery of its presence in appreciable (pharmacologically significant) concentrations in red wines, which was upheld as an explanation for the socalled “French Paradox” (5, 6), where despite a diet rich in fat and alcohol, the expected increase in coronary heart disease among the wine-drinking French population is not observed. Consequently, a series of sensitive chromatographic methods have been developed to quantify its concentration in red wines from various regions (710). To date, 1 has been shown to exhibit an impressive array of antioxidative (11), anticarcinogenic (12), antiinflammatory (13), and cardioprotective (5) properties. For example, studies have shown that 1 inhibits the initiation, promotion, and progression of cancer (13), inhibits platelet aggregation and coagulation (14), and acts as a vasorelaxant by inhibiting the contractile response of the aorta to noradrenaline (15). The various relevant physiological properties of 1 have been reviewed and summarized in considerable depth (16). Despite the enthusiastic push toward reaching clearer assessment of resveratrol’s potential health protecting properties in humans, studies of its in vivo metabolism * Tel: 351-22-9866104. Fax: [email protected].

351-22-9866192. E-mail:

Figure 1. Chemical structure of resveratrol 1.

have left doubts remaining as to the exact nature of its metabolic profile. Although O-conjugation of structurally related polyhydroxylic substrates, such as for example the flavanoids, with glucuronic acid is known to represent a major metabolic pathway for such compounds (17), it was somewhat surprising therefore to fail to discover suitable literature precedent for the chemical synthesis of the regioisomeric 3-O-β-D- and 4′-O-β-D-glucuronide conjugates (2 and 3, respectively) (Figure 2) of 1 for unequivocal identification and quantification of these conjugates in preclinical metabolic studies. Indeed, two distinct O-glucuronide conjugates were detected in a bioavailability study of 1 in the rat, but it was not possible to elucidate the exact position of conjugation or clearly ascertain the relative extent of conjugation at each position. Consequently, enzymatic deconjugation with a β-glucuronidase was required to liberate free 1 for more exact quantification (18). Although chemical synthesis of the naturally occurring 3-O-β-glycoside (piceid) of 1 has been previously reported (19, 20), two drawbacks of this synthesis are apparent from the outset. The traditional Wittig phosphorus ylid chemistry employed therein to install the central carbon-carbon double bond gave predominantly what would be in this case, the unwanted cis-isomer (cis/trans, 2.3:1 ratio), and equally unfortunately, chromatographic separation of the cis/trans geometric isomers was difficult, contributing to diminish the overall yield of the desired trans-stilbene even

10.1021/bc020048x CCC: $25.00 © 2003 American Chemical Society Published on Web 12/06/2002

Bioconjugate Chem., Vol. 14, No. 1, 2003 263

Figure 2. Chemical structures of resveratrol-3-O-β-D-glucuronide 2 and resveratrol-4′-O-β-D-glucuronide 3. Table 1. Analytical Data (Combustion Analyses, New Compounds) no.

formula

calculated % (C, H)

found % (C, H)

6 8 3 10 11 13 2

C19H21IO10 C31H32O14 C20H20O9 C19H21IO11 C21H23IO12 C31H32O14 C20H20O9

C, 42.55; H, 3.95 C, 59.23; H, 5.13 C, 59.40; H, 4.98 C, 41.32; H, 3.83 C, 42.44; H, 3.90 C, 59.23; H, 5.13 C, 59.40; H, 4.98

C, 42.85; H, 3.95 C, 59.03; H, 4.82 C, 59.12; H, 5.29 C, 41.76; H, 4.08 C, 42.86; H, 4.08 C, 59.23; H, 5.15 C, 59.09; H, 5.26

further. Second, the deprotection procedure described for cleavage of a methoxyl protecting group in the last step (large excess of sodium ethanethiolate in DMF at 140 °C for 10 h) was considered incompatible with the presence of the rather sensitive O-β-glucuronide substituent. Elsewhere, despite the considerable number of reported methods for chemical synthesis of 1 itself and related derivatives (21-24), there has appeared to date only a single recent report of enzymatic glucuronidation of 1 and its cis-isomer using human liver microsomes (25). Unfortunately, this enzymatic method allows for the preparation of only very low quantities of the required glucuronide conjugates, and this lack of appropriate methodology rendered necessary the development of efficient regio- and stereoselective chemical synthesis of both the 3-O-β-D- and 4′-O-β-D-glucuronide conjugates of 1 using a strategy based on a novel Heck coupling of iodoarylO-β-D-glucuronate esters with appropriately substituted styrenes. 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 internal standard, and data are reported in the following order: chemical shift (ppm), multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; dd, double doublet; br, broad), number of protons, and approximate coupling constant in Hertz. IR spectra were measured with a Bomem Hartmann & Braun MB Series FTIR spectrometer using KBr tablets (see Table 1. Analytical HPLC was performed on an automated Agilent 1100 Series HPLC, using LiChrospher 100 RP-18 (5µm) LiChroCart 250-4 Cartridges (Merck) in combination with acetonitrile/water (1% formic acid) 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 done 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. 3,5-Diacetoxystyrene 7 was prepared as previously reported by Guiso et al (26) from commercially available 3,5-dihydroxybenzaldehyde. 4-Ac-

etoxystyrene 12 was purchased from Aldrich and used as received. 3,5-Dihydroxyiodobenzene 9 was prepared from commercially available 3,5-dimethoxyaniline via Sandmeyer reaction (59% yield) and demethylation (57% HI, 90% yield) as previously described by Deboves et al (27). The trichloroacetimidate 5 was prepared according to the procedure of Fischer et al. (28). 4-Iodo-O-2,3,4-triacetyl-β-D-glucuronopyranosidobenzene Methyl Ester (6). To a stirred and cooled (icewater bath) solution of 4-iodophenol 4 (0.66 g, 2.98 mmol) and trichloroacetimidate 5 (2.0 g, 4.18 mmol) in dry dichloromethane (20 mL) under argon was added anhydrous boron trifluoride diethyl etherate (0.59 g, 4.18 mmol) dropwise. The resulting solution was allowed to stir at room temperature for 1 h and then poured onto ice-water (30 mL). The phases were separated, and the aqueous phase was extracted by dichloromethane (10 mL). The combined organic layers were washed by water (40 mL) and brine (40 mL), dried over anhydrous magnesium sulfate, filtered, and evaporated (40 °C, water aspirator pressure) to leave a pale orange residue that solidified on standing. Recrystallization (dichloromethane/ 2-propanol) afforded colorless crystals (1.30 g, 66%) of mp 161-162 °C. Anal. (C19H21IO10) C,H. IR (KBr) 1756 cm-1 (broad, CdO). 1 H NMR (CDCl3) δ 7.61 (d, 2H, J ) 8.9 Hz), 6.79 (d, 2H, J ) 8.9 Hz), 5.40-5.25 (m, 3H), 5.12 (d, 1H, J ) 7.3 Hz), 4.18 (m, 1H), 3.75 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H). 13C NMR (CDCl3) δ 170.7, 169.9, 169.8, 167.3, 157.1, 139.1, 119.9, 99.5, 73.2, 72.2, 71.6, 69.5, 53.6, 21.2, 21.1. (E)-1-(3,5-Diacetoxyphenyl)-2-(4′-O-2,3,4-triacetylβ-D-glucuronopyranosidophenyl)ethene Methyl Ester (8). To a stirred solution of 3,5-diacetoxystyrene 7 (0.31 g, 1.42 mmol) and the iodophenyl-O-β-D-glucuronate ester 6 (1.14 g, 1.74 mmol) in dimethylformamide (15 mL) at room temperature under argon were added benzyltriethylammonium chloride (0.32 g, 1.42 mmol), tributylamine (0.69 g, 3.73 mmol), and palladium (II) acetate (15.3 mg, 0.068 mmol). The resulting pale orange solution was stirred at 110 °C for 30 min and then allowed to cool to room temperature. The mixture was poured onto water (50 mL) with stirring causing formation of a pale yellow precipitate, which was filtered off and washed with water (5 mL). The precipitate was dissolved in dichloromethane (20 mL), and the organic layer was washed by water (20 mL), 2N HCl (10 mL), water again (10 mL), and brine (20 mL), then dried over anhydrous magnesium sulfate, filtered, and evaporated (40 °C, water aspirator pressure) to leave an orange/brown foam. Chromatography over silica gel (petroleum ether/ethyl acetate, 1:1) afforded the major product as an off-white solid that was triturated with diethyl ether to afford a white powder (0.49 g, 55%) of mp 152-154 °C. Anal. (C31H32O14) C,H. IR (KBr) 1759 cm-1 (broad, CdO). 1 H NMR (DMSO-d6) δ 7.58 (d, 2H, J ) 8.7 Hz), 7.29 (d, 1H, J ) 16.4 Hz), 7.26 (d, 2H, J ) 2.1 Hz), 7.14 (d, 1H, J ) 16.4 Hz), 7.02 (d, 2H, J ) 8.7 Hz), 6.88 (t, 1H, J

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) 2.1 Hz), 5.7 (d, 1H, J ) 8.0 Hz), 5.47 (t, 1H, J ) 9.7 Hz), 5.11 (dd, 1H, J ) 8.0 and 9.7 Hz), 5.07 (t, 1H, J ) 9.7 Hz), 4.72 (d, 1H, J ) 9.7 Hz), 3.64 (s, 3H), 2.28 (s, 6H), 2.02 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H). 13C NMR (DMSO-d6) δ 170.6, 170.4, 170.1, 170.1, 168.1, 157.1, 152.2, 140.5, 132.6, 130.6, 129.2, 126.6, 118.0, 117.6, 97.8, 72.1, 72.0, 71.5, 70.0, 53.7, 21.8, 21.3, 21.2. (E)-1-(3,5-Dihydroxyphenyl)-2-(4′-O-β- D -glucuronopyranosidophenyl)ethene (3), (trans-Resveratrol-4′-O-β-D-glucuronide). To a stirred and cooled (ice-water bath) suspension of the glucuronate ester 8 (0.39 g, 0.61 mmol) in methanol (9 mL) was added dropwise aqueous 1 N sodium hydroxide solution (9 mL, 9 mmol). The resulting mixture was stirred in the cold for 30 min and then at room temperature for 15 min. The resulting solution was diluted with water (6 mL), and sufficient Amberlyst 15 ion-exchange resin was added with stirring to maintain pH ∼2-3. The resin was then filtered off and washed with water (3 mL). The combined filtrate was evaporated (60 °C, water aspirator pressure). Toluene (5 mL) was added to the residue and reevaporated. The residue was dried at room temperature over phosphorus pentoxide under high vacuum overnight. The resulting solid was triturated with diethyl ether (3 mL) and filtered to give a pale beige solid (0.21 g, 84%), which decomposed without melting above 230 °C. Anal. (C20H20O9) C,H. IR (KBr) 3383 (very broad, OH) and 1730 cm-1 (Cd O). 1 H NMR (DMSO-d6) δ 9.22 (br s, 2H), 7.52 (d, 2H, J ) 8.7 Hz), 7.00 (d, 2H, J ) 8.7 Hz), 6.99 (d, 1H, J ) 16.4 Hz), 6.92 (d, 1H, J ) 16.4 Hz), 6.41 (d, 2H, J ) 1.7 Hz), 6.13 (t, 1H, J ) 1.7 Hz), 5.46 (d, 1H, J ) 3.75 Hz), 5.25 (br, 1H), 5.07 (d, 1H, J ) 7.5 Hz), 3.90 (d, 1H, J ) 9.6 Hz). 13C NMR (DMSO-d6) δ 171.2, 159.5, 157.5, 140.0, 132.1, 128.7, 128.3, 128.3, 117.3, 105.5, 103.1, 100.8, 76.8, 76.4, 74.0, 72.4. 3-Hydroxy-5-iodo-O-2,3,4-triacetyl-β- D -glucuronopyranosidobenzene Methyl Ester (10). To a stirred and cooled (ice-water bath) solution of 3,5dihydroxyiodobenezene 9 (2.36 g, 10 mmol) and the trichloroacetimidate 5 (4.79 g, 10 mmol) in dry dichloromethane (50 mL) under argon was added boron trifluoride diethyl etherate (1.42 g, 10 mmol) dropwise. The resulting mixture was allowed to stir at room temperature for forty minutes and then poured onto ice-water (30 mL). The phases were separated and the aqueous phase was extracted by dichloromethane (10 mL). The combined organic layers were washed by water (40 mL) and brine (40 mL), dried over anhydrous magnesium sulfate, filtered, and evaporated (40 °C, water aspirator pressure) to leave a pale yellow foam. Chromatography over silica gel (petroleum ether/ethyl acetate 2:1) afforded the major product as a white solid, (1.93 g, 35%) of mp 186-188 °C. Anal. (C19H21IO11) C,H. IR (KBr) 3332 (OH) and 1748 cm-1 (broad, CdO). 1 H NMR (CDCl3) δ 6.97 (dd, 1H, J ) 1.4 and 2.1 Hz), 6.91 (dd, 1H, J ) 1.4 and 2.1 Hz), 6.53 (t, 1H, J ) 2.1 Hz), 6.23 (br, 1H), 5.45-5.20 (m, 3H), 5.15 (d, 1H, J ) 7.5 Hz), 4.25 (d, 1H, J ) 9.6 Hz), 3.76 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H). 13C NMR (CDCl3) δ 171.0, 170.4, 169.9, 167.7, 158.3, 158.0, 120.8, 119.2, 105.3, 99.4, 94.3, 73.0, 72.3, 71.5, 69.7, 53.7, 21.2, 21.2, 21.1. 3-Acetoxy-5-iodo-O-2,3,4-triacetyl-β-D-glucuronopyranosidobenzene methyl ester (11) To a cooled (ice-water bath) stirred solution of the phenol 10 (1.81 g, 3.28 mmol) in dry dichloromethane (50 mL) was added pyridine (0.39 g, 4.92 mmol), 4-(dimethylamino)pyridine (10 mg) followed by acetic anhydride

(0.50 g, 4.92 mmol) dropwise. The resulting mixture was stirred in the cold for 30 min and then washed by 2N HCl (20 mL), water (20 mL), and brine (20 mL), then dried over anhydrous magnesium sulfate, filtered, and evaporated (40 °C, water aspirator pressure). Toluene (15 mL) was added to the residue and re-evaporated. The residue was recrystallized (dichloromethane/petroleum ether) to give a white solid, (1.63 g, 83%) of mp 126-128 °C. Anal. (C21H23IO12) C,H. IR (KBr) 1757 cm-1 (broad, CdO). 1 H NMR (CDCl3) δ 7.25 (dd, 1H, J ) 1.4 and 2.1 Hz), 7.21 (dd, 1H, J ) 1.4 and 2.1 Hz), 6.75 (t, 1H, J ) 2.1 Hz), 5.45 (m, 3H), 5.15 (d, 1H, J ) 7.0 Hz), 4.22 (d, 1H, J ) 9.1 Hz), 3.75 (s, 3H), 2.30 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H). 13C NMR (CDCl3) δ 170.6, 169.9, 169.8, 169.4, 167.3, 157.8, 152.0, 126.7, 124.2, 111.4, 99.3, 93.6, 73.2, 72.2, 71.5, 69.4, 53.6, 21.6, 21.2, 21.1. (E)-1-(3-Acetoxy-5-O-2,3,4-triacetyl-β- D -glucuronopyranosidophenyl)-2-(4′- acetoxyphenyl)ethene Methyl Ester (13). To a stirred solution of the iodophenyl-O-β-D-glucuronate ester 11 (1.60 g, 2.69 mmol) and 4-acetoxystyrene 12 (0.40 g, 2.45 mmol) in dimethylformamide (40 mL) at room temperature under argon were added benzyltriethylammonium chloride (0.56 g, 2.45 mmol), tributylamine (1.19 g, 6.44 mmol), and palladium (II) acetate (27 mg, 0.122 mmol). The resulting pale orange solution was stirred at 120 °C for 40 min and then allowed to cool to room temperature. The mixture was poured onto water (50 mL) and extracted by dichloromethane (2 × 30 mL). The organic extracts were washed by 2N HCl (20 mL), water (20 mL), and brine (20 mL), dried over anhydrous magnesium sulfate, filtered, and evaporated (40 °C, water aspirator pressure) to leave a brown oil. Chromatography over silica gel (petroleum ether/ethyl acetate, 2:1) afforded the major product as a pale yellow foam that was recrystallized (dichloromethane/diethyl ether) to give a white solid (0.69 g, 45%) of mp 153-154 °C. Anal. (C31H32O14) C,H. IR (KBr) 1761 cm-1 (broad, CdO). 1 H NMR (CDCl3) δ 7.51 (d, 2H, J ) 8.7), 7.11 (d, 2H, J ) 8.7 Hz), 7.07 (d, 1H, J ) 16.4 Hz), 7.01 (m, 1H), 6.99 (m, 1H), 6.97 (d, 1H, J ) 16.4 Hz), 6.66 (t, 1H, J ) 2.1 Hz), 5.45-5.25 (m, 3H), 5.21 (d, 1H, J ) 7.2 Hz), 4.24 (d, 1H, J ) 9.2 Hz), 3.75 (s, 3H), 2.33 (s, 3H), 2.33 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H). 13C NMR (CDCl3) δ 170.7, 170.0, 169.9, 169.8, 169.8, 167.4, 158.0, 152.2, 151.0, 140.3, 135.0, 130.1, 128.2, 127.9, 122.5, 115.2, 113.3, 110.2, 99.5, 73.3, 72.3, 71.6, 69.5, 53.6, 21.7, 21.2, 21.2, 21.1. (E)-1-(5-Hydroxy-3-O-β-D-glucuronopyranosidophenyl)-2-(4′-hydroxyphenyl)ethene (2), (trans-resveratrol-3-O-β-D-glucuronide). To a stirred and cooled (icewater bath) suspension of the glucuronate ester 13 (0.56 g, 0.89 mmol) in methanol (13 mL) was added dropwise aqueous 1 N sodium hydroxide solution (13 mL, 13 mmol). The resulting mixture was stirred in the cold for 30 min and then at room temperature for 40 min. The mixture was then gently warmed to 45 °C for 30 min to aid dissolution and then stirred at room temperature for a further hour. The resulting solution was diluted with water (12 mL), and sufficient Amberlyst 15 ion-exchange resin was added with stirring to maintain pH ∼2-3. The resin was then filtered off and washed with water (10 mL). The combined filtrate was evaporated (60 °C, water aspirator pressure). Toluene (10 mL) was added to the residue and re-evaporated. The residue was dried at room temperature over phosphorus pentoxide under high vacuum overnight. The resulting solid was triturated with diethyl ether (3 mL) and filtered to give a pale beige

Bioconjugate Chem., Vol. 14, No. 1, 2003 265 Scheme 1. Synthesis of Resveratrol-4′-O-β-D-glucuronide

3a

a Reagents: (a) BF .OEt , CH Cl , 0 °C to room temperature, 1h, (66%); (b) Pd(OAc) , BnEt N+Cl-, Bu N, DMF, 110 °C, 30 min, 3 2 2 2 2 3 3 (55%); (c) (i) 1N NaOH (aq), MeOH, 0 °C, 30 min, (ii) Amberlyst 15 ion-exchange resin (84%).

solid (0.33 g, 92%), which decomposed without melting above 210 °C. Anal. (C20H20O9) C,H. IR (KBr) 3372 (very broad, OH) and 1749 cm-1 (Cd O). 1 H NMR (DMSO-d6) δ 9.54 (br, 2H), 7.41 (d, 2H, J ) 8.5 Hz), 7.02 (d, 1H, J ) 16.4 Hz), 6.87 (d, 1H, J ) 16.4 Hz), 6.76 (d, 2H, J ) 8.5 Hz), 6.66 (t, 1H), 6.58 (t, 1H), 6.32 (t, 1H), 4.99 (d, 1H, J ) 7.3 Hz), 3.89 (d, 1H, J ) 9.6 Hz), 3.40 (t, 1H, J ) 9.1 Hz), 3.31 (t, 1H, J ) 8.7 Hz), 3.25 (t, 1H, J ) 8.4 Hz). 13C NMR (DMSO-d6) δ 171.2, 159.5, 159.4, 158.4, 140.4, 129.6, 129.0, 126.1, 116.6, 108.2, 105.8, 103.6, 101.1, 76.8, 76.5, 74.0, 72.4. RESULTS AND DISCUSSION

It has been previously shown that O-glucuronidation of 1 is a major metabolic pathway in the rat, giving rise to two O-conjugated glucuronide metabolites, which unfortunately could not be unambiguously characterized (18). Of course, O-glucuronidation of 1 is certainly expected to represent a major metabolic pathway in other species, including humans and hence synthesis of the individually pure O-glucuronide standards would greatly assist metabolic and pharmacokinetic studies of 1. Although a number of chemical methods exist for preparation of O-glucuronide conjugates (29), synthesis of the individual resveratrol 3-O-β-D- and 4′-O-β-D-conjugates 2 and 3 represents a particular challenge, due to the presence of three equally reactive hydroxyl groups shared between two phenyl rings, separated by a highly conjugated central carbon-carbon double bond, structural features which effectively combine to complicate many protection/deprotection strategies. As such, direct methods of selective mono-O-glucuronidation of 1 were not considered. Alternatively, a convergent strategy was devised, where the two phenyl constituents of the stilbenic skeleton of glucuronides 2 and 3 were to be constructed separately, i.e a phenyl ring of the molecule containing the acetyl protected O-β-D-glucuronide moiety and an appropriately O-substituted styrene, giving rise to two suitably functionalized synthons which could then theoretically be cross-coupled via a palladium catalyzed olefinic arylation, more commonly known as the Heck reaction (30, 31). Starting with synthesis of resveratrol-4′-O-β-D-glucuronide 3, preference for the use of the aryl iodide was based on generally enhanced reactivity over bromides in the Heck reaction thereby also providing the possibility to employ phosphine-free conditions. Accordingly, the

reaction of commercially available 4-iodophenol 4 with Schmidt’s trichloroacetimidate 5 proceeded smoothly under boron trifluoride catalysis to give in a completely stereoselective fashion (exclusive β-anomer formation) the acetyl protected O-β-D-glucuronate ester 6 in good yield (66%). Preparation of the required diacetoxy olefin 7 was achieved as described (26) without incident. This olefin was in fact used in a Heck-mediated synthesis of 1 with 4-acetoxyiodobenzene (26), although when investigating this same reaction in this laboratory, the long reaction time and conditions (17 h at reflux) caused extensive deacetylation and decomposition, and the overall yield of 1 was rather low. In accordance, initial attempts to couple the glucuronate ester 6 with the olefin 7 under similar Heck conditions (Pd(OAc)2, Et3N or Bu3N, DMF or CH3CN, 100-120 °C) were frankly disappointing, giving rise to mostly decomposition products (possibly again through extensive deacetylation) and eventually furnishing the desired protected stilbene derivative 8 in rather unsatisfactory yields (12-20%). The use of tetraalkylammonium halides (in particular chlorides) has been found to significantly accelerate certain Heck reactions (32) (Jeffery modification), and it was therefore extremely gratifying to find that the addition of one molar equivalent of benzyltriethylammonium chloride (used instead of the standard additive, tetrabutylammonium chloride) relative to the olefin greatly improved the situation; in fact, after only 30 min at 110 °C, the reaction had proceeded cleanly, and the desired protected (E)stilbene 8 could be obtained exclusively in considerably improved yield (55%). To the best of the author’s knowledge, this is the first reported example of the successful use of this type of aryl-O-β-D-glucuronate ester in a Heck coupling reaction. Thereafter, deprotection of the acetyl protecting groups of the both the glucuronide and phenolic hydroxyl groups was easily achieved using aqueous 1 N sodium hydroxide in methanol. Acidic workup (Amberlyst 15 ion-exchange resin) and evaporation of the solvents afforded resveratrol-4-O-β-D-glucuronide 3 in both good yield (84%) and excellent purity (HPLC, >99%) with minimum purification necessary (Scheme 1). Thus, for the synthesis of resveratrol-3-O-β-D-glucuronide 2, 3,5-dihydroxyiodobenzene 9 and commercially available 4-acetoxystyrene 12 were considered suitably convenient starting materials. Aryl iodide 9 was prepared from commercially available 3,5-dimethoxyaniline as previously described (27) (attempts to convert another readily available precursor, 3,5-dimethoxychlorobenzene,

266 Bioconjugate Chem., Vol. 14, No. 1, 2003 Scheme 2. nide 2a

Synthesis of Resveratrol-3-O-β-D-glucuro-

will be found useful for the synthesis of O-β-D-glucuronide conjugates of related polyhydroxy stilbenes. ACKNOWLEDGMENT

The author wishes to thank Ana Paula Freitas for providing 1H, 13C NMR spectra and microanalytical data. LITERATURE CITED

a Reagents: (a) 5, BF .OEt , CH Cl , 0 °C to room tempera3 2 2 2 ture, 40 min (35%); (b) Ac2O, pyridine, DMAP, CH2Cl2, 0°C, 30 min, (83%); (c) Pd(OAc)2, BnEt3N+Cl-, Bu3N, DMF, 120 °C, 40 min, (45%); (d) (i) 1N NaOH (aq), MeOH, 0 °C for 30 min, room temperature for 40 min, 45 °C for 30 min, (ii) Amberlyst 15 ionexchange resin (92%).

into the corresponding iodide by reaction with magnesium and iodine gave inconsistent results in agreement with observations made by another group (33)). Attempted mono-acetylation of 9 (1 equiv Ac2O, Py, DMAP, CH2Cl2) gave a three-component mixture, containing predominantly diacetylated product and starting material rather than the desired mono-acetate. More encouraging however, was the completely stereoselective reaction of 9 with 1 equiv of Schmidt’s trichloroacetimidate 5, again under boron trifluoride catalysis, which allowed isolation of the mono-O-β-D-glucuronate ester 10 in moderate yield (35%). Some unreacted 9 (∼25%) could be recovered by chromatography and recycled. Since the presence of free hydroxyl groups is preferably avoided in the Heck reaction, 10 was O-acetylated to give fully protected 11. Heck coupling of 11 with the styrene 12 under similar conditions used beforehand (Pd(OAc)2, Bu3N, BnEt3NCl, DMF, 120 °C) proceeded rapidly (40 min) and without significant decomposition, affording exclusively the protected (E)-stilbene 13 in now quite acceptable yield (45%). Subsequent acetyl-group deprotection of 13 was achieved under similar basic conditions as before, and the free glucuronide 2 could be isolated in excellent yield (92%) and purity (>99% homogeneous by HPLC) again with minimal purification (Scheme 2). CONCLUSIONS

Short, efficient chemical methods suitable for the stereo- and regioselective, multimilligram to gram scale synthesis of highly pure individual 3-O-β-D- and 4′-O-βD-glucuronide conjugates 2 and 3 of resveratrol 1 have been developed, based on a novel Heck coupling of iodoaryl-O-β-D-glucuronate esters with O-protected styrenes. These compounds will find use in the more accurate determination of the metabolic and pharmacokinetic profile of 1 in several species, including humans. It is further hoped that the methodology herein described

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