Concise and Scalable Synthesis of Aspalathin, a ... - ACS Publications

Dec 19, 2013 - Copyright © 2013 The American Chemical Society and American Society of ... Ana R. Jesus , Ana P. Marques , Amélia P. Rauter. Pure and...
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Concise and Scalable Synthesis of Aspalathin, a Powerful Plasma Sugar-Lowering Natural Product Ze Han,† Matthew C. Achilonu,† Pravin S. Kendrekar,† Elizabeth Joubert,‡ Daneel Ferreira,§ Susan L. Bonnet,† and Jan H. van der Westhuizen*,† †

Department of Chemistry, University of the Free State, Nelson Mandela Avenue, Bloemfontein, 9301, South Africa ARC Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch, 7599, South Africa § Department of Pharmacognosy and Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United States ‡

S Supporting Information *

ABSTRACT: Aspalathin (1), a dihydrochalcone C-glucoside, exhibits powerful plasma sugar-lowering properties and thus potentially could be used to treat diabetes. Small quantities occur in rooibos tea, manufactured via fermentation of the leaves of Aspalathus linearis, hence necessitating the need for an efficient and concise synthesis. Efforts to synthesize aspalathin (1) via coupling of a glucose donor to the nucleophilic phloroglucinol ring of the dihydrochalcone moiety have invariably failed, presumably because of ring deactivation by the electron-withdrawing carbonyl group. Reduction of the carbonyl group of a chalcone (15) and coupling of the resulting 1,3diarylpropane (16) to tetra-O-benzyl-β-D-glucopyranose afforded the Cglucosyl-1,3-diarylpropane (17). Regiospecific benzylic oxidation regenerated the carbonyl group and afforded the per-O-methylaspalathin (1a) quantitatively. This method was not successful with the per-O-benzylprotected dihydrochalcone. However, the nucleophilicity of the phenolic hydroxy groups of the dihydrochalcone or its acetophenone precursor is not diminished by the carbonyl group. Thus, glucosylation of the di-O-benzylacetophenone (5c) at −40 °C afforded the α-O-glucoside (19) in 86% yield. Raising the temperature allowed facile BF3-catalyzed rearrangement to the β-C-glucoside (6b), which upon hydrogenation, afforded aspalathin (1) in 80% overall yield [based on the usage of di-O-benzylphloroacetophenone (5c) and tetra-O-benzyl-1α-fluoro-βD-glucose (2e)].

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aspalathin in human urine and plasma samples after ingestion of aspalathin (1)10 suggests that the aspalathin core structure is not changed during metabolism. The detection of the unconjugated aspalathin in the urine of pigs11 after ingestion of an aspalathin-enriched rooibos extract indicated at least limited absorption and bioavailability. These observations have stimulated renewed interest in aspalathin, also as a nutraceutical ingredient in foods and beverages.12,13 The low natural occurrence of aspalathin (1) in the leaves of A. linearis and its innate susceptibility to oxidative conversion significantly complicate isolation and purification via chromatography. After fermentation, only about 7% of the aspalathin (1) in fresh A. linearis leaves remains in the commercial product.14 Such insufficient quantities to optimally explore the interesting biological properties of aspalathin prompted efforts by Minehan and co-workers15 to develop a synthesis route toward the natural product.

ooibos is a slightly sweet and mildly astringent fragrant herbal tea produced via fermentation of the commercially cultivated leaves and twigs of the endemic South African plant Aspalathus linearis (Burm.f.) Dahlg. (Fabaceae). Its caffeine-free and low-tannin status and potential health-promoting properties, notably its antioxidant activity, have stimulated its popularity and consumption. Rooibos extracts are also used by the beverage, food, and nutraceutical industries for its flavor and antioxidant properties.1 Aspalathin (1), a dihydrochalcone C-glucopyranoside, was first characterized by Koeppen and co-workers2 in 1965. It occurs exclusively in the leaves of A. linearis (rooibos) and is the major secondary metabolite, possessing antioxidant3−5 and antimutagenic6 properties. Recent studies7,8 indicate that aspalathin has beneficial effects on glucose homeostasis in type 2 diabetes through stimulating glucose uptake in muscle tissues and insulin secretion from pancreatic β-cells. Phenols are metabolized by liver enzymes, leading to reduced bioavailability and to high levels of conjugates in the plasma and urine.9 The detection of aspalathin glucuronyl and sulfate conjugates in rat liver extracts and O-linked methyl-, sulfate-, glucuronide-, and O-methyl-O-glucuronide derivatives of © 2013 American Chemical Society and American Society of Pharmacognosy

Special Issue: Special Issue in Honor of Otto Sticher Received: October 9, 2013 Published: December 19, 2013 583

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The aforementioned problems regarding the supply of aspalathin (1) from natural sources necessitated the need for the development of a concise and industrial-scalable synthesis for aspalathin (1) as well as some analogues. Aldol condensation of the per-O-methylacetophenone (5b) and benzaldehyde (7b) afforded the per-O-methylchalcone (15) (Scheme 3). Catalytic hydrogenation of 15 under high pressure (30 MPa) yielded the 1,3-diarylpropane (16). Coupling of 16 with tetra-O-benzylglucose (2c), using TFAA preactivation,16 afforded the glucosylated 1,3-diarylpropane (17), the latter three conversions occurring in near quantitative yields. Oxidation of 17 with DDQ in moist CH2Cl2−1,3dioxane (1.5 equiv of H2O as oxygen source) afforded the anticipated dihydrochalcone-C-glucoside (1a) in 83% overall yield. Efforts to obtain underivatized aspalathin (1) via removal of the methyl protecting groups using published methods (e.g., BBr3 ), 17 however, gave poor yields. Coupling of the dihydrochalcone (18), obtained via oxidation of the diarylpropane (16), with tetra-O-benzylglucose (2c) was unsuccessful, hence further supporting the notion that the carbonyl group deactivates the aromatic phloroglucinol-type ring (Scheme 3). Our one-pot synthesis of compound 16, a natural product from the roots of Litsea hypophaea,18 is an improvement on the synthesis of Drumm et al.,19 which required a two-step reduction of 15 to form 16. This work also represents the first synthesis of per-O-methyldihydrochalcone (18, 99% yield), a natural product isolated from the roots of L. hypophaea by Wang et al.20 The NMR spectra of 17 were recorded at 140 °C to eliminate the adverse effects of rotational isomerism due to hindered rotation about the C-glucosidic bond. Salient in the 1 H NMR spectra were the three 1,3-diarylpropane methylene moieties that resonated as three sets of diastereotopic proton multiplets at δH 2.63 (H-1, m), 2.57 (H-3, t, J = 7.5 Hz), and 1.80 (H-2, m), the aromatic H-5′′ singlet at δH 6.48, and the anomeric doublet at δH 4.72 (J = 9.8 Hz), diagnostic of a C-

Efforts15 to synthesize aspalathin (1), via a process involving the chalcone intermediate 8a (Scheme 1), failed. In addition, Cglucosylation of acetophenone (5a) afforded the C-glucosyl aryl ketone intermediate (6a) in trace amounts. This is presumably due to deactivation of the aromatic nucleophile by the electronwithdrawing carbonyl group. Such reduced nucleophilicity is presumably further compounded by complexation of the Lewis acid catalyst with the carbonyl oxygen. The C-arylglucoside (4a), devoid of the carbonyl group, was obtained in high yield from 2a and 3a, but efforts to transform 4a via Friedel−Crafts acylation into 6a also failed, presumably due to removal of the benzyl protecting groups by the Lewis acid. Attempts to obtain 10 via the Vilsmeier−Haack reaction of 4a similarly were unsuccessful. Minehan15 subsequently synthesized aspalathin in eight steps and a ca. 20% overall yield via formation of the C-arylglucoside (4b) to afford the glucosylated benzaldehyde (10). Homologation of 10 using alkyne 11 and the Bestman−Ohira reagent afforded alkynol 12. Subsequent benzylic oxidation and deprotection via catalytic hydrogenation afforded aspalathin (1) in 20% yield (Scheme 2).

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RESULTS AND DISCUSSION Herein, we report our results on the synthesis of aspalathin (1), based on the hypothesis that the deactivating carbonyl group of the benzyl-protected acetophenones 5a and 5b inhibits their Cglucosylation.

Scheme 1. Minehan’s15 Unsuccessful Aldol-Type Synthesis of Aspalathin (1)

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Scheme 2. Minehan’s15 Successful Synthesis of Aspalathin (1)

Scheme 3. Synthesis of O-Methyl-Protected Aspalathin (1a) via the Diarylpropane Intermediate (16)a

5′ of the B-ring, confirming the C−C coupling of the glucosyl moiety. Efforts to obtain aspalathin (1) via the O-methoxymethyl and O-benzyl equivalents of 16 (Scheme 3) failed. The MOM groups did not survive the glucose coupling conditions, while the benzyl groups did not survive the benzylic oxidation conditions. The electron-withdrawing effect of the carbonyl group, however, does not reduce the nucleophilicity of the phenolic hydroxy group of di-O-benzylphloroacetophenone (5c), as treatment of 5c with glucose donor 2a, 2d, or 2e yielded the relevant O-glucoside (19) in 53%, 66%, and 84% yields, respectively (Scheme 4). The best yield (84%) was obtained with BF3-catalyzed coupling of the α-fluoroglucopyranoside 2e at −40 °C. In our hands, tranformation of 2a to 2e utilizing published methods21 gave yields of less than 20%. Synthesis of 2e via the imidate 2d, however, afforded 2e in near quantitative yield (98%). In contrast with the anomeric β-configuration (3J1,2 = 9.8 Hz) of the C-glucosyl moiety of aspalathin (1), the O-glucoside (19) possesses an anomeric α-configuration (3J1,2 = 3.5 Hz). No evidence of the β-anomer was found by NMR spectroscopy in the crude reaction mixture. Salient is the absence of hindered rotation about the O-glucosidic bond of 19, attributed to an increased distance between the bulky acetophenone and sugar moieties. Well-resolved NMR spectra were thus obtained at room temperature. The anomeric C-1′ and H-1′ atoms were deshielded by the acetal oxygens and resonated at δC 96.8 and δH 5.43, permitting differentiation from the corresponding Cglucoside (6b) with its ether-type anomeric center inducing resonances at δC 73.6 and δH 4.73, respectively. The aromatic acetophenone protons (H-3 and H-5) resonated as two doublets (J = 2.0 Hz) at δH 6.42 and 6.29. Upon treating the O-glucoside (19) with BF3·OEt2 at −15 °C, the C-glucoside (6b), previously obtained by Kumazawa et al.22 during the synthesis of C-glucosylflavones, was obtained in good yield via rearrangement of the O-glucoside to the ortho Cglucoside.23 Acquiring the NMR spectrum of the C-glucoside 6b required, as for the C-glucosides 17 and 1a, a temperature of 140 °C in DMSO-d6 to overcome the adverse effects of

a Reagents and reaction conditions: (a) HOAc, TFAA, CH2Cl2, −20 °C; (b) 50% KOH, 18-crown ether, 1,4-dioxane, 60 °C; (c) 20% Pd(OH)2/C, H2 (30 MPa), EtOAc−EtOH; (d) BF3·OEt2, TFAA, MeCN; (e) DDQ−H2O, CH2Cl2−1,4-dioxane, 0 °C, 12 h.

glucoside with β-configuration. The 13C NMR spectrum was characterized by the anomeric C-coupled carbon at δC 73.6 that correlated with the anomeric proton (δH 4.72) in an HSQC experiment. Acquisition of the 1H NMR spectra of 1a also required an elevated temperature (140 °C in DMSO-d6) to induce free rotation about the C-glucosidic bond. The CH2−CH2 moiety resonated as two multiplets at δH 3.05 and 2.93 in the 1H NMR spectrum and the carbonyl carbon at δC 198.4 in the 13C NMR spectrum. The anomeric proton resonated as a doublet at δH 4.73 and correlated with the anomeric carbon at δC 73.6 in the edited HSQC spectrum. The 9.8 Hz coupling constant confirmed a β-configured C-glucosidic bond. The one-proton singlet at δH 6.52 in the 1H NMR spectrum corresponded to H585

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Scheme 4. Synthesis of Aspalathin (1) via an O-Glucosidic Intermediatea

a Reagents and conditions: (a) BF3·OEt2−CH2Cl2; −78 °C → −40 °C; (b) −40 °C → −15 °C; (c) 50% NaOH, 1,4-dioxane, 40 °C; (d) Pd(OH)2/ C.

suppliers and were used without further purification. All solvents used for routine isolation of products and chromatography were reagent grade. Air- and moisture-sensitive reactions were performed under an argon atmosphere. Flash column chromatography was performed using silica gel of 230−400 mesh with the indicated solvents. TLC was performed using 0.25 mm silica gel plates. IR spectra were recorded on an FT-IR spectrometer. The 1H and 13C NMR spectra were recorded on a Bruker Avance spectrometer (600 MHz) in CDCl3 (δH = 7.24; δC = 77.2), methanol-d4 (δH = 4.87 and 3.31; δC = 49.2), and DMSO-d6 (δH = 2.50; δC = 39.5). Chemical shifts (δ) are reported as ppm on the δ-scale, and coupling constants (J) were measured in Hz. High-resolution mass spectra were recorded on a Waters Micromass LCT Premier TOF-MS mass spectrometer. All samples were dissolved and diluted to 2 ng/μL and infused without additives. Syntheses. 1-O-Acetyl-2,3,4,6-tetra-O-benzyl-β-D-glucopyranose (2a). A mixture of 2,3,4,6-tetra-O-benzyl-β-D-glucopyranose (2c) (3.7 mmol; 2.00 g), pyridine (48 mmol; 5 mL), and Ac2O (112 mmol; 10 mL) was stirred at room temperature for 24 h. The reaction was quenched with crushed ice, filtered, and washed with cold H2O (3 × 200 mL), and the precipitate was dissolved in CH2Cl2 (200 mL). The CH2Cl2 solution was dried over MgSO4 and concentrated under vacuum to yield 2a as a colorless oil (2.62 g, 14.8 mmol; 100%). The spectroscopic data corresponded to the literature data.21 2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl-1-O-trichloroacetimidate (2d). Trichloroacetonitrile (74.04 mmol; 7.7 mL) was added to a solution of 2,3,4,6-tetra-O-benzyl-β-D-glucopyranose (2c) (14.81 mmol; 8.00 g) in anhydrous CH2Cl2 (40 mL) under argon. After stirring at room temperature for 2−3 min, the mixture was cooled to −78 °C and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (1.5 mmol; 0.23 mL) was added. The reaction mixture was stirred for 30 min, the temperature raised to −30 °C, and the solution stirred for an additional hour. The reaction mixture was flashed through Celite (3 × 5 cm column), washed with CH2Cl2 (2 × 50 mL), dried over MgSO4, and concentrated under vacuum to yield 2d as a light brown oil (9.41 g, 14.37 mmol; 97%). TLC showed two predominant spots for the αand β-anomers, and the crude 2d was used as starting material for the synthesis of 2e without further purification. The spectroscopic data corresponded to reported values.25 2,3,4,6-Tetra-O-benzyl-1α-fluoro-β-D-glucopyranose (2e). The dried, crude mixture of α- and β-anomers 2d (2.6 g, 4.0 mmol) was dissolved in anhydrous CH2Cl2 (45 mL) in a polyethylene container under argon. The mixture was stirred for 5 min and cooled to −30 °C, and 70% HF−pyridine (127.36 mmol; 9.4 mL) was added. After stirring for 30 min, the temperature of the mixture was allowed to rise to 0 °C, where it was stirred for a further 6 h. The mixture was transferred to a 1 L polyethylene container, and a slurry of silica gel (10−15 g) in CH2Cl2 (200 mL) was added slowly. The resulting mixture was filtered, dried over MgSO4, concentrated under vacuum,

rotational isomerism. The anomeric ethereal proton resonates as a doublet at δH 4.73 that correlated with the anomeric carbon at δC 73.6 in the edited HSQC spectrum. The 9.8 Hz coupling constant indicated a β-anomeric C-glucosidic bond. The anomeric configuration thus changed from α to β during conversion from an O- to a C-glucoside. A NOESY correlation between H-5 (δH 6.43) and the methylene resonances of two aromatic benzyl protecting groups (δH 5.27 and 5.18, respectively) confirmed the formation of the ortho-C-glucoside via an O → C inter- or intramolecular glucosidic rearrangement.23 Upon treatment of a mixture of 6b and 3,4-dibenzyloxybenzaldehyde (7a) with 50% NaOH, the chalcone 8b was obtained in 96% yield and proved to be identical to the compound described by Kumazawa et al.22 On catalytic hydrogenation of 8b, aspalathin (1), with spectroscopic data identical to published data,24 was obtained as the sole product in 99% yield (Scheme 4). In subsequent syntheses, the O-glucoside (19) was not isolated and 6b was prepared according to the Kumazawa procedure.22 The reaction mixture comprising 2e (1 equiv), 5c (2 equiv), and BF3·OEt2 (2 equiv) was monitored at −40 °C by TLC until the glucosyl donor 2e was fully consumed, i.e., when O-coupling was completed. The temperature was subsequently gradually increased to −15 °C. Upon completion of the O−C rearrangement (TLC), a small quantity of H2O was added rapidly at −15 °C to destroy the BF3·OEt2, and 6b was obtained in a yield of 84%. Attempts to synthesize aspalathin without such accurate temperature control gave only trace amounts of the synthetic target. The potential of the scalability of the process was recently demonstrated by the synthesis of a 15 g sample of aspalathin. We have thus developed efficient syntheses of free phenolic and O-methyl-protected aspalathin in 80% and 83% overall yields, respectively, based on protocols to circumvent the electron-withdrawing effect of the benzylic carbonyl group with concomitant lowering of the nucleophilicity of the aromatic methine carbons. The overall yield of free phenolic aspalathin (1) is based on usage of the sugar fluoride (2e) and di-O-benzyl phloroacetophenone (5c) as starting materials.

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EXPERIMENTAL SECTION

General Experimental Procedures. Unless noted otherwise, all starting materials and reagents were obtained from commercial 586

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and purified with silica flash-chromatography (n-hexane−CDCl3− EtOAc, 8:1:1) to yield 2e as white crystals from n-hexane−EtOAc (2.13 g, 3.93 mmol, 98%), mp 38−40 °C. The spectroscopic data corresponded to reported values.21 2,4-Di-O-benzyl-6-hydroxyacetophenone (5c). Anhydrous 2,4,6trihydroxyacetophenone (1.0 g, 5.95 mmol) and anhydrous K2CO3 (2.50 g, 20 mmol) in dry DMF (6 mL) were stirred at room temperature under argon for 30 min. The mixture was cooled to 0 °C, and benzyl chloride (1.65 mL; 14.3 mmol) was added. The mixture was allowed to warm to room temperature and stirred until complete conversion of the starting material and monobenzylated intermediate [TLC (n-hexane−EtOAc, 8:2), Rf 0.12 and 0.26, respectively]. The mixture was quenched with 10% NH4Cl (100 mL) and extracted with Et2O (3 × 100 mL), and the ether layers were dried over MgSO4 and concentrated under vacuum to yield the crude product (2.44 g). Preparative-TLC (n-hexane−EtOAc, 8:2) yielded 5c as white crystals from n-hexane−EtOAc (2.03 g, 5.82 mmol, 98%, Rf 0.56), mp 108− 109 °C. The spectroscopic data corresponded to literature values.22 4 , 6 - D i b e n z y l o x y - 2 - O - ( 2 ′ , 3 ′ , 4 ′ , 6 ′ - t e t r a - O - b e n z y l -α - D glucopyranosyl)acetophenone (19). BF3·Et2O (0.15 mL, 1.16 mmol) was added dropwise to a stirred mixture of the acetophenone (5c) (522 mg, 1.5 mmol), the 2-fluoroglucose (2e) (542 mg, 1 mmol), and powdered 4 Å molecular sieves (600 mg) in CH2Cl2 (10 mL) at −78 °C. After stirring for 30 min, the temperature was allowed to gradually rise to −40 °C. Stirring was continued for 1 h, when TLC indicated disappearance of the 2-fluoroglucose (2e) and the formation of the Oglucoside (19). The reaction mixture was quenched with H2O and filtered through a Celite pad. The filtrate was extracted with CHCl3 (3 × 20 mL), the organic layer dried over anhydrous MgSO4, and the solvent evaporated under reduced pressure. The resulting syrup was purified by silica gel flash chromatography (n-hexane−EtOAc, 5:1) to give 19 as a viscous, colorless oil (749 mg, 0.86 mmol, 86%): IR (neat) νmax 2596, 1732, 1602, 1183, 1075 cm−1; 1H NMR (600 MHz, DMSO-d6, 140 °C) δ 7.15−7.60 (30H, m, aromatic H), 6.43 (1H, d, J = 2 Hz, H-3), 6.24 (1H, d, J = 2 Hz, H-5), 5.43 (1H, d, J = 3.8 Hz, H-1′), 4.55−5.05 (12H, m, benzylic H), 4.07 (1H, t, J = 9.8 Hz, H-3′), 3.88 − 3.91 (1H, m, H-5′), 3.78 (1H, dd, J = 2.0, 9.8 Hz, H6′a), 3.67 (1H, dd, J = 4.0, 9.8 Hz, H-4′), 3.63 (1H, dd, J = 4.0, 9.8 Hz, H-2′), 3.58 (1H, dd, J = 2.0, 9.8 Hz, H-6′b), 2.55 (3H, s); 13C NMR (150 MHz, DMSO-d6, 140 °C) δC 201.0 (CO), 156.9 (3C, Oaromatic), 115.9−138.6 (24C, aromatic), 96.8 (C-1′), 95.5, (C-3), 94.7 (C-5), 81.6 (C-3′), 79.7 (C-2′), 75.7 (C-4′), 75.0 (C-1), 73.4, 73.2, 70.5, 70.2 (4C, benzylic OCH2), 71.3 (C-5′), 68.2 (C-6′), 32.7 (CO-CH3); HRESIMS m/z = 893.3632 [M + Na] (calcd for C56H54O9, 893.3666). 4,6-Dibenzyloxy-3-C-(2′,3′,4′,6′-tetra-O-benzyl-β-D-glucopyranosyl)-2-hydroxyacetophenone (6b). BF3·Et2O (1.43 mL, 11.6 mmol) was added dropwise to a stirred mixture of 4,6-dibenzyloxy-2hydroxyacetophenone (5c) (3.0 g, 8.7 mmol), 2,3,4,6-tetra-O-benzylD-glucopyranosyl fluoride (2e) (3.14 g, 5.79 mmol), and powdered 4 Å molecular sieves (6.0 g) in CH2Cl2 (60 mL) at −78 °C. The mixture was stirred for 30 min, and the temperature was subsequently allowed to gradually increase to −40 °C. Stirring was continued for 1 h, when TLC indicated the disappearance of fluoride 2e and the formation of the O-glucoside (19), and subsequently at −15 °C for 4 h. The reaction mixture was quenched with H2O and filtered through a Celite pad. The filtrate was extracted with CHCl3 (3 × 20 mL), the organic layer dried over anhydrous MgSO4, and the solvent evaporated under reduced pressure. The resulting syrup was purified by silica gel flash chromatography (n-hexane−EtOAc, 5:1) to yield 6b as a pale green oil (4.24 g, 4.86 mmol, 84%). The spectroscopic data corresponded to literature values.22 3,4,4′,6′-Tetrabenzyloxy-2′-hydroxy-3′-C-(2″,3″,4″,6″-tetra-Obenzyl-β-D-glucopyranosyl)chalcone (8b). A solution of 6b (1.74 g, 2 mmol), 3,4-dibenzyloxybenzaldehyde (7a) (763 mg, 2.4 mmol) in 1,4dioxane (20 mL), and 50% aqueous NaOH (10 mL) was stirred at 40 °C for 20 h. The mixture was acidified with 2 M HCl (100 mL) and extracted with EtOAc (2 × 50 mL). The combined extracts were washed with H2O and brine, dried over MgSO4, and evaporated in vacuo. The residue was purified by flash column chromatography on

silica gel (n-hexane−EtOAc, 7:1) to give 8b as a viscous yellow oil (2.25 g, 1.92 mmol, 96%). The spectroscopic data correspond to literature values.22 3,4,2′,4′,6′-Pentahydroxy-3-C′-(2″,3″,4″,6″-tetrahydroxy-β-Dglucopyranosyl)dihydrochalcone (Aspalathin) (1). A suspension of 4,6-dibenzyloxy-3-C-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)-2-hydroxyacetophenone (8b) (1.17 g, 1 mmol) and 20% Pd(OH)2/C (130 mg) in EtOAc (5 mL)−MeOH (10 mL) was stirred under H2 at atmospheric pressure for 48 h. The reaction mixture was filtered, concentrated under a vacuum, and recrystallized from acetone to yield 1 as white crystals (447 mg, 0.99 mmol; 99%), mp 148.6 °C, lit. mp24 148−150 °C. The spectroscopic data corresponded to the published values.24 (E)-2,4,6,3′,4′-Pentamethoxychalcone (15). To a solution of 2,4,6trimethoxyacetophenone (5b) (2.00 g, 9.5 mmol) in 1,4-dioxane (10 mL) were added 3,4-dimethoxybenzaldehyde (7b) (1.92 g, 4.67 mmol) and 50% aqueous KOH (10 mL). The mixture was refluxed for 14 h, quenched with ice-cold H2O (50 mL), acidified with 10% HCl (20 mL), and extracted with EtOAc (3 × 100 mL). The organic layer was washed with H2O and brine and dried over anhydrous MgSO4, and the solvent was evaporated under reduced pressure. The resulting syrup was chromatographed on a silica gel column (Et2O−EtOAc, 9:1) to yield the protected chalcone 15 as a yellow powder (3.38 g, 9.41 mmol, 99%). The spectroscopic data corresponded to reported values.26 2,4,6,3′,4′-Pentamethoxy-1,3-diarylpropane (16). A solution of 3,4,2′,4′,6′-pentamethoxychalcone (15) (1.85 g, 5 mmol), 20% Pd(OH)2/C (200 mg) in CH2Cl2 (10 mL), and EtOH (10 mL) was stirred under H2 (30 MPa) for 24 h. The reaction mixture was filtered, concentrated, and recrystallized from EtOAc to yield 16 as white crystals (1.77 g, 4.95 mmol; 99%), mp 88.3 °C, lit. mp18 88−89 °C. The spectroscopic data corresponded to reported data.18 2′′,4′′,6′′,3′,4′-Pentamethoxy-3-(2′′′,3′′′,4′′′,6′′′-tetra-O-benzylβ-D-C-glucopyranosyl)-1,3-diarylpropane (17). 2,3,4,6-Tetra-O-benzyl-β-D-glucopyranose (2c) (1.08 g; 2 mmol) was stirred in TFAA (2 mL; 14.4 mmol)−MeCN (20 mL) at room temperature for 20 min followed by addition of the 1,3-diarylpropane 16 (865 mg; 2.5 mmol) in CH2Cl2 (2 mL). The mixture was cooled to −30 °C before BF3· OEt2 (0.65 mL; 5 mmol) was added. The temperature of the mixture was raised to 0 °C, and the mixture stirred for 1 h. The reaction was quenched with H2O (1 mL), filtered, and extracted with EtOAc (3 × 50 mL). The organic phases were combined, dried over MgSO4, and concentrated under a vacuum. The resulting syrup was chromatographed on a silica gel column (n-hexane−CH2Cl2−EtOAc, 10:1:1) to yield compound 17 as a colorless oil (1.62 g, 1.84 mmol; 92%): IR (neat) νmax 2934, 1731, 1600, 1064 cm−1; 1H NMR (600 MHz, DMSO-d6, 140 °C) δH 7.10−7.40 (20H, m, aromatic), 6.85 (1H, dd, J = 2.2, 8.5 Hz, H-6′), 6.77 (1H, d, J = 2.2 Hz, H-2′), 6.65 (1H, d, J = 8.5 Hz, H-5′), 6.48 (1H, s, H-5′′), 4.73 (1H, d, J = 9.8 Hz, H-1‴), 4.01−4.95 (8H, benzylic H), 4.37 (1H, t, J = 9.8 Hz, H-2‴), 3.50−3.80 (15H, ArOCH3), 3.69 (1H, d, J = 9.8 Hz, H-3‴), 3.63 (2H, m, H-6‴), 3.60 (1H, t, J = 9.8 Hz, H-4‴), 3.53 (m, H-5‴), 2.63 (m, H-1), 2.57 (1H, t, J = 7.5 Hz, H-3), 1.80 (m, H-2); 13C NMR (150 MHz, DMSOd6, 140 °C) δC 159.8 (C-2′′, C-4′′, C-6′′), 150.2 (C-3′), 148.5 (C-4′), 114.3−139.6 (24C, Ar-C), 121.2 (C-5′), 115.0 (C-2′), 114.7 (C-6′), 95.5 (C-5′′), 87.3 (C-3‴), 80.6 (C-2‴), 79.5 (C-4‴), 79.4 (C-5‴), 74.2 (C-1‴), 70.0−80.1 (8C, benzylic OCH2), 62.7 (C-6‴), 56.5−57.2 (5C, ArOCH3), 35.4 (C-3), 31.5 (C-2), 23.4 (C-1); HRESIMS m/z 891.4068 [M + Na]+ (calcd for C54H60O10 891.4084). 2″,3″,4″,6″-Tetra-O-benzyl-3,4,2′,4′,6′-penta-O-methylaspalathin (1a). A mixture of 17 (1.40 g; 1.6 mmol), DDQ (3.68 g; 16 mmol) in CH2Cl2 (30 mL), and 1,4-dioxane (15 mL) was added to H2O (0.1 mL; 5.5 mmol) at 0 °C. The reaction mixture was stirred for 12 h, diluted with EtOAc (100 mL), and washed with a saturated NaHCO3 solution (3 × 100 mL) and subsequently with H2O (3 × 100 mL). The organic phases were combined, dried over MgSO4, concentrated under a vacuum, and purified by flash chromatography (n-hexane−CH2Cl2−EtOAc, 5:1:1) to give 1a as a colorless oil (1.31 g, 1.47 mmol; 92%): 587

dx.doi.org/10.1021/np4008443 | J. Nat. Prod. 2014, 77, 583−588

Journal of Natural Products

Article

IR (neat) νmax 2938, 1731, 1672, 1129 cm−1; 1H NMR (600 MHz, DMSO-d6, 140 °C) δ 6.82−7.60 (20H, m, aromatic), 6.52 (1H, s, H5′), 4.55−4.90 (8H, m, benzylic Hs), 4.73 (1H, d, J = 9.8 Hz H-1″), 4.38 (1H, t, J = 9.8 Hz, H-2″), 3.75−3.95 (15H, ArOCH3), 3.76 (2H, m, H-6″), 3.72 (1H, d, J = 9.8 Hz, H-3″), 3.63 (1H, t, J = 9.8 Hz, H4″), 3.60 (1H, m, H-5″), 3.05 (1H, Hα), 2.93 (1H, Hβ); 13C NMR (150 MHz, DMSO-d6, 140 °C) δC 198.4 (CO), 159.7, 154.3, 149.9, 139.7, 139.3 (aromatic, 5C), 113.0−139.3 (31C, aromatic), 95.5 (C5′), 87.3 (C-3″), 80.6 (C-2″), 79.5 (C-4″), 79.4 (C-5″), 73.6 (C-1″), 74.6, 74.2, 73.2, 70.6 (4C benzylic, OCH2), 62.9 (C-6′′), 56.5−57.3 (5C, ArOCH3), 38.8 (Cα), 19.7 (Cβ); HRESIMS m/z 905.3868 [M + Na]+ (calcd for C54H58O11 905.3877). 3-(3,4-Dimethoxyphenyl)-1-(2,4,6-trimethoxyphenyl)propan-1one (18). A mixture of 2′,4′,6′,3″,4″-pentamethoxy-1,3-diarylpropane (16) (550 mg; 1.6 mmol) and DDQ (3.68 g; 16 mmol) in CH2Cl2 (30 mL)−1,4- dioxane (15 mL) was added to H2O (0.1 mL; 5.5 mmol) at 0 °C. The reaction mixture was stirred for 12 h, diluted with EtOAc (100 mL), and washed with saturated NaHCO3 solution (3 × 100 mL) and subsequently with H2O (3 × 100 mL). The organic phases were combined, dried over MgSO4, concentrated under vacuum, and purified by flash chromatography (n-hexane−CH2Cl2−EtOAc, 5:1:1) to yield 18 as a colorless oil (570 mg, 1.47 mmol; 99%). The spectroscopic data corresponded to reported values.20

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of all new compounds are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +27-51-401-2782. Fax: +27-51-444-6384. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support by the South African Medical Research Council, the South African Agricultural Research Council, the South African National Research Foundation THRIP, and the University of the Free State, Bloemfontein, South Africa, is gratefully acknowledged.

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DEDICATION Dedicated to Prof. Dr. Otto Sticher, of ETH-Zurich, Zurich, Switzerland, for his pioneering work in pharmacognosy and phytochemistry

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