Hybrid of Resveratrol and Glucosamine: An Approach To Enhance

Article Cite This: Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Hybrid of Resveratrol and Glucosamine: An Approach To Enhance Antioxidant Effect against DNA Oxidation Liang-Liang Bao and Zai-Qun Liu* Department of Organic Chemistry, College of Chemistry, Jilin University, Changchun 130021, China

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S Supporting Information *

ABSTRACT: Resveratrol exhibits various pharmacological activities, which are dependent upon phenolic hydroxyl groups. In this work, glucosamine, lipoic acid, or adamantanamine moiety was applied for attaching to ortho-position of hydroxyl group in resorcinol moiety of resveratrol (known as position-2). Antioxidant effects of the obtained hybrids were characterized using DNA oxidative systems mediated by •OH, Cu2+/ glutathione (GSH), and 2,2′-azobis(2-amidinopropanehydrochloride) (AAPH), respectively. The glucosyl-appended imine and amine at position-2 of resveratrol were found to show higher inhibitory effects than other resveratrol derivatives against AAPH-induced DNA oxidation. The antioxidative effect was quantitatively expressed by stoichiometric factor (n, the number of radical-propagation terminated by one molecule of antioxidant). The stoichiometric factors of glucosyl-appended imine and amine of resveratrol increased to 4.74 (for imine) and 4.97 (for amine), respectively, higher than that of resveratrol (3.70) and glucoside of resveratrol (3.49). It was thereby concluded that the combination of resveratrol with glucosamine at position-2 represented a novel pathway for modifying resveratrol structure in the protection of DNA against peroxyl radical-mediated oxidation.

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INTRODUCTION Resveratrol (3,4′,5-trihydroxystilbene) was found to be active in the chemoprevention of cancer,1 and growing evidence indicated that resveratrol was also effective in numerous disease models including cardiovascular disease,2 diabetes,3 neurodegenerative disease,4 cancer,5 etc. Resveratrol acted as a structural moiety of polyphenolic oligomers in plants6 and as a basic scaffold in the discovery of novel drugs.7 However, resveratrol might be a prooxidant to induce oxidative breakage of cellular DNA in the presence of metal ions.8 Therefore, burgeoning numbers of resveratrol derivatives were currently found in the literature with the intensive goal of medicinal application.9 The biological activity of resveratrol was related to its hydroxyl groups, which were able to trap free radicals in vivo.10 In the preparation of resveratrol derivatives, both HeckHiyama cross-coupling11 and Wittig reaction12 provided with powerful strategies for achieving resveratrol derivatives with various substituents attaching to the stilbene scaffold. As shown in Figure 1, a typical resveratrol derivative was produced by the substitution of one benzene ring in resveratrol by bicyclo[1.1.1]-pentane13 or deferiprone,14 or the CC bond in resveratrol was replaced by multiple CC bonds,15 1,4,2-dioxazole,16 or selenophene.17 A combination of two pharmacological molecules was a popularly used strategy to harvest duplicate functions within a single drug, in which 4′position in resveratrol usually bridged with other medicinal © XXXX American Chemical Society

moieties via the esterification with 4′-hydroxyl group or amidation with the 4′-amino group (replacing the 4′-hydroxyl group by amino group in advance). For example, aspirin,18 caffeic acid,19 p-dimethylaminophenol,20 and tacrine21 attached to the 4′-position in resveratrol, which formed hybrids with multiple pharmacological activities. In addition, some efforts contributed to attaching sugar moieties to hydroxyl groups in resveratrol (see Figure 1),22 owing to resveratrol glycoside possessing high bioavailability.23 However, there has still been scanty reports to-date on revealing the bioactivity of glucosylappended imine or amine of resveratrol.24 In light of the recognition on the correlation of DNA damage with diseases,25 we attempted to test the influence of appended moieties on inhibitory effects of resveratrol derivatives against DNA oxidative damage, which was mediated by •OH, Cu2+/glutathione (GSH), and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH, R-NN-R, R = −CMe2C(NH) NH2), respectively, because •OH was known as an important member in the family of reactive oxygen species (ROS), while the decomposition of AAPH provided with peroxyl radical (ROO•) to mimic DNA undergoing peroxidation in vivo. The combination of Cu(II) with glutathione in vivo might be harmful to health because of Received: May 26, 2018

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DOI: 10.1021/acs.chemrestox.8b00136 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology

Figure 1. Recently published resveratrol derivatives.

the generation of glutathione radical (GS•) via the oxidation of GSH by Cu(II).

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mmol 1 (256 mg) and 10 mmol TBDMSCl (1.51 g); then 10 mmol imidazole (680 mg dissolved in 4 mL of THF) was added under ice bath and stirred for 10 h. After the reaction mixture was concentrated under reduced pressure, the residue was purified by silica gel column chromatography with petroleum ether as eluent to give (E)-2,4di(tert-butyldimethylsilyl)oxy-6-(4′-(tert-butyldimethylsilyl)oxylstyryl) benzaldehyde (1a) (430 mg, yield 72%) as a green oil. Then −CHO in the aforementioned compound (1a) was reduced by NaBH4 to afford −CH2OH. To 5 mL of THF solution of 1a (120 mg, 0.2 mmol) was added NaBH4 (7.6 mg, 0.2 mmol) and stirred at 0 °C for 20 min, followed by the addition of 0.1 mL of water and stirring for 5 min. The aqueous solution of NH4Cl (54 mg dissolved in 0.3 mL of water) was added and stirred for 20 min and then diluted with THF. After the solid in the suspension was removed, the liquid phase was concentrated under reduced pressure, and crude product was purified by silica gel column chromatography with petroleum ether/ ethyl acetate mixture (v/v = 20:1) as eluent to give (E)-2,4-di(tertbutyldimethylsilyl)oxy-6-(4′-(tert-butyldimethylsilyl)oxylstyryl)benzyl alcohol (2a) as a colorless oil in a yield of 69% (83 mg). Finally, the protective groups (TBDMS) at hydroxyl groups were removed under acidic condition. To 3 mL of THF solution of 2a (36 mg, 0.06 mmol) was added 200 μL of trifluoroacetic acid and stirred at 0 °C for 5 h in the atmosphere of N2. After the organic media were evaporated under reduced pressure, the residue was purified by silica gel column chromatography with ethyl acetate/methanol mixture (v/v = 10:1) as eluent to give 2 as a brown oil in a yield of 71% (11 mg). 1H NMR (400 MHz, CD3OD): δ 7.29 (d, J = 8.3 Hz, 2H), 7.22 (d, J = 16.1 Hz, 1H), 6.81 (d, J = 16.1 Hz, 1H), 6.67 (d, J = 7.9 Hz, 2H), 6.14 (s, 1H), 6.05 (s, 1H), 4.48 (s, 2H). 13C NMR (101 MHz, CDCl3): δ 155.9, 155.5, 154.9, 139.9, 131.2, 130.3, 129.7, 126.6, 122.7, 119.8, 113.9, 110.2, 58.0. HRMS (ESI): calcd for [M + H]+ of C15H15O4, 259.0970; found, 259.2835. (E)-2,4-Dihydroxy-6-(4′-hydroxystyryl)benzyl 5-(1,2-dithiolan-3yl)pentanoate (3). The esterification of −CH2OH in 3 with lipoic acid was carried out with 2a being the starting material. To 10 mL of

MATERIALS AND METHODS

Materials and Instrumentation. 2,2′-Azobis(2-amidinopropane hydrochloride) (AAPH) and naked DNA sodium salt were products of ACROS Organics, Geel, Belgium, and used as received. Solvents and reagents used in the synthesis were obtained commercially and used as such unless noted otherwise. All of the products were identified by 1H and 13C NMR spectroscopy (Bruker Avance III 400 MHz spectrometer) that were outlined in the Supporting Information. The molecular weight was detected by high resolution mass spectra (HRMS) equipped with ESI as the ionization mode (Agilent 1290micrOTOF Q II). Synthesis of Resveratrol Derivatives. (E)-2,4-Dihydroxyl-6-(4′hydroxylstyryl)benzaldehyde (1). The formylation of resveratrol was carried out in 60 mL of acetonitrile solution of resveratrol (456 mg, 2 mmol), to which 2 mmol N,N-dimethylformamide (DMF, 146 mg) and 2.3 mmol POC13 (352 mg) were added at −5 °C and stirred for 3 h at the same temperature. After the produced solid was collected and washed with cold acetonitrile, 30 mL of water was added and heated at 55 °C under stirring for 2 h. Then the solid was collected and washed with cold water. After dried over reduced pressure, 1 was obtained as yellowish solid in a yield of 62% (320 mg). MP 209 °C. 1 H NMR (400 MHz, DMSO-d 6): δ 10.27 (s, 1H), 7.69 (d, J = 16.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 16.0 Hz, 1H), 6.79 (d, J = 7.9 Hz, 2H), 6.62 (s, 1H), 6.22 (s, 1H). 13C NMR (101 MHz, CD3OD): δ 192.9, 165.5, 157.8, 145.6, 134.6, 128.2, 127.4, 119.6, 115.2, 111.6, 106.1, 104.4, 100.9. HRMS (ESI): calcd for [M + H] + of C15H13O4, 257.0814; found, 257.3239. (E)-2,4-Dihydroxyl-6-(4′-hydroxylstyryl)benzyl alcohol (2). Before −CHO in 1 was reduced to form −CH2OH, phenolic hydroxyl groups in 1 were protected by tert-butyldimethylsilyl chloride (TBDMSCl). To 5 mL of tetrahydrofuran (THF) was added 1 B


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1H), 5.31 (d, J = 5.0 Hz, 1H), 5.12 (d, J = 4.6 Hz, 1H), 5.05 (d, J = 5.2 Hz, 1H), 4.81 (d, J = 7.5 Hz, 1H), 4.66 (t, J = 5.6 Hz, 1H), 3.74 (dd, J = 10.7, 4.8 Hz, 1H), 3.50 (dt, J = 11.7, 5.9 Hz, 1H), 3.25 (m, 4H). 13C NMR (101 MHz, DMSO-d6): δ 159.3, 158.8, 157.8, 139.8, 128.9, 128.4, 125.7, 115.9, 107.6, 105.2, 103.2, 101.1, 77.6, 77.2, 73.7, 70.2, 61.2. N-2,4-Dihydroxy-6-((E)-4-hydroxystyryl)benzylidene-2-deoxy-αD-glucosamine (6). The reaction of D-glucosamine with 1 was capable of furnishing 6 directly. To 5 mL of stirred aqueous solution of D-glucosamine (179 mg, 1 mmol) was added 256 mg of 1 (1 mmol, dissolved in 6 mL of THF) and one drop of formic acid. The reaction mixture was stirred at room temperature for 24 h. The solvent was removed in vacuo, and the crude product was purified by thin layer chromatography with ethyl acetate/methanol mixture (v/v = 25:1) as eluent to afford 6 in a yield of 6% (25 mg). 1H NMR (400 MHz, DMSO-d6): δ 8.54 (s, 1H), 7.42 (d, J = 8.6 Hz, 2H), 7.29 (d, J = 16.0 Hz, 1H), 6.86 (d, J = 16.0 Hz, 1H), 6.77 (d, J = 8.5 Hz, 2H), 6.57 (d, J = 2.3 Hz, 1H), 6.24 (d, J = 2.3 Hz, 1H), 5.21 (d, J = 3.5 Hz, 1H), 3.98 (q, J = 7.1 Hz, 1H), 3.66−3.56 (m, 2H), 3.52 (dd, J = 11.7, 5.1 Hz, 1H), 3.19 (t, J = 9.4 Hz, 1H), 2.91 (dd, J = 10.5, 3.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6): δ 171.8, 159.3, 159.1, 157.5, 148.7, 139.9, 132.4, 128.8, 122.4, 115.9, 107.3, 105.2, 102.1, 89.1, 72.4, 70.2, 69.9, 60.6, 54.5. HRMS (ESI): calcd for [M + H]+ of C21H24NO8, 418.1502; found, 418.4623. N-2,4-Dihydroxy-6-((E)-4-hydroxystyryl)benzyl-2-deoxy-D-glucosamine (7). To 2 mL of stirred methanolic solution of acetylappended glucosamine (173 mg, 0.5 mmol) was added 1.5 mL of methanolic solution of 1a (299 mg, 0.5 mmol) and one drop of formic acid, and the stirring was continued at room temperature for 2.5 h to achieve imine (1,3,4,6-tetra-O-acetyl-2-N-2,4-di(tertbutyldimethylsilyl)oxy-6-((E)-4′-(tert-butyldimethylsilyl)oxystyryl)benzylidene-2-deoxy-β-D-glucosamine, 6a) as green solid in a yield of 58% (270 mg). To 3 mL of stirred THF solution of 6a (232 mg, 0.25 mmol) was added 19 mg of NaBH4 (0.5 mmol). The mixture was stirred at 0 °C for 20 min, and 0.1 mL of water was added and stirred for 5 min. After an aqueous solution of NH4Cl (54 mg dissolved in 0.3 mL of water) was added and stirred for 20 min, the solution was evaporated under reduced pressure. The ethyl acetate and water were added to dissolve the residue, and the organic layer was collected and dried with MgSO4. After the organic solvent was evaporated, 1,3,4,6tetra-O-acetyl-2-N-(2,4-di(tert-butyldimethylsilyl)oxy-6-((E)-4′-(tertbutyldimethylsilyl)oxystyryl)benzyl)-2-deoxy-D-glucopyranose (7a) was achieved as brown oil in a yield of 44% (102 mg). To 2 mL of vigorously stirred methanolic solution of 7a (93 mg, 0.1 mmol) was added 69 mg of K2CO3 (0.5 mmol), and the stirring was continued at room temperature for 6 h, followed by the addition of 0.5 mL of trifluoroacetic acid and stirring for 2 h. The solvent was removed in vacuo, and the crude product was purified by thin layer chromatography with ethyl acetate/methanol mixture (v/v = 3:1) as eluent to give 7 as yellowish oil in a yield of 36% (15 mg). 1H NMR (400 MHz, DMSO-d6): δ 7.37 (d, J = 8.5 Hz, 2H), 7.08 (d, J = 16.3 Hz, 1H), 7.01 (d, J = 15.9 Hz, 1H), 6.84 (d, J = 8.5 Hz, 2H), 6.78 (d, J = 15.9 Hz, 1H), 6.75 (d, J = 16.3 Hz, 1H), 6.54 (d, J = 2.2 Hz, 1H), 6.51 (d, J = 2.5 Hz, 1H), 6.31 (d, J = 2.2 Hz, 1H), 6.30 (d, J = 2.5 Hz, 1H), 5.38 (d, J = 3.3 Hz, 1H), 5.06 (t, J = 9.6 Hz, 1H), 4.38−2.87 (10H), 1.29−1.22 (4H). • OH- and Cu2+/GSH-Induced Oxidations of DNA. DNA was dissolved in phosphate buffered solution (PBS1: 6.1 mM Na2HPO4, 3.9 mM NaH2PO4) to reach 2.24 mg/mL as the stock solution, tetrachlorohydroquinone (TCHQ) was dissolved in dimethyl sulfoxide (DMSO) to reach 120 mM as the stock solution, and H2O2 was diluted in PBS1 to reach 30.0 mM as the stock solution. To 13.4 mL of DNA solution was added 1.0 mL of H2O2, 0.5 mL of TCHQ solution, and 0.1 mL resveratrol derivatives (dissolved in DMSO as the stock solution). The aforementioned mixture was aliquoted into test tubes, with each containing 2.0 mL (the final concentrations of DNA, H2O2, TCHQ, and resveratrol derivatives were 2.00 mg/mL, 1.5, 3.0 mM, and 200 μM, respectively. The same volume of DMSO was contained in the control experiment to eliminate the influence from DMSO). The test tubes were incubated

CH2Cl2 were added 206 mg of lipoic acid (1 mmol), 237 mg of 1,3dicyclohexylcarbodiimide (DCC, 1.15 mmol), and 12.2 mg of 4dimethylaminopyridine (DMAP, 0.1 mmol) and stirred at 0 °C for 15 min, followed by the addition of 5 mL of CH2Cl2 solution of 2a (600 mg, 1 mmol) and stirring for 15 min at the same temperature. The solution was allowed to increase to room temperature and stirred for 18 h. After the reaction mixture was washed with water (3 × 50 mL), the organic layer was dried with MgSO4, filtered, and evaporated. The residue was purified by flash chromatography with petroleum ether/ ethyl acetate mixture (v/v = 20:1) as eluent to afford 3a as yellowish oil in a yield of 38% (300 mg). 1H NMR (400 MHz, CD3OD): δ 7.45 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 15.6 Hz, 1H), 6.96 (d, J = 16.2 Hz, 1H), 6.87 (d, J = 8.0 Hz, 2H), 6.59 (s, 1H), 6.33 (s, 1H), 5.32 (s, 1H), 3.43 (m, 1H), 3.13 (m, 2H), 2.33 (m, 3H), 1.62 (m, 7H), 1.03 (s, 27H), 0.28 (s, 18H). Following the same procedure as removing the protective group in 2a, 3a (197 mg, 0.25 mmol) and 500 μL of trifluoroacetic acid in 3 mL of THF furnished 3 as a brown oil in a yield of 23% (26 mg). 1H NMR (400 MHz, CDCl3): δ 7.39 (d, J = 8.5 Hz, 1H), 7.16 (d, J = 16.0 Hz, 1H), 6.90 (d, J = 16.1 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H), 6.76 (d, J = 2.1 Hz, 1H), 6.30 (d, J = 2.1 Hz, 1H), 5.28 (s, 1H), 3.51 (dd, J = 14.0, 6.9 Hz, 1H), 3.13 (ddd, J = 17.8, 11.6, 4.9 Hz, 2H), 2.48−2.28 (m, 3H), 1.94−1.80 (m, 1H), 1.73−1.52 (m, 6H). 13C NMR (101 MHz, CDCl3): δ 173.7, 156.6, 155.9, 155.7, 140.5, 131.1, 130.6, 127.9, 123.6, 120.4, 117.1, 110.5, 109.7, 56.2, 40.2, 38.5, 34.6, 34.2, 28.7, 24.8, 18.3. HRMS (ESI): calcd for [M + H]+ of C23H27O5S2, 447.1300; found, 447.1755. 2-((Adamantan-1-ylamino)methyl) resveratrol (4). The adamantan-1-yl group was directly furnished by the reaction of 1 with 1adamantanamine, followed by the reduction with NaBH4. To 8 mL of methanol was dissolved 151 mg of 1-adamantanamine (1 mmol) and 256 mg of 1 (1 mmol), as well as one drop of formic acid, and the stirring was continued at room temperature overnight. The solvent was removed to afford imine as brown oil, which was diluted with methanol and cooled in ice bath. Then 38 mg of NaBH4 (1 mmol) was added and stirred for 20 min, and three drops of water was added and stirred for 5 min. After an aqueous solution of NH4Cl (162 mg dissolved in 1.5 mL of water) was added and stirred for 20 min, the solution was evaporated under reduced pressure. The ethyl acetate and water were added to dissolve the residue, and the organic layer was collected and dried with MgSO4. After the organic solvent was evaporated, 4 was obtained as brown solid in a yield of 17% (69 mg). MP 218 °C. 1H NMR (400 MHz, CD3OD): δ 7.41 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 16.1 Hz, 1H), 6.93 (d, J = 16.1 Hz, 1H), 6.79 (d, J = 8.6 Hz, 2H), 6.62 (d, J = 2.3 Hz, 1H), 6.27 (d, J = 2.3 Hz, 1H), 4.76 (s, 2H), 2.18 (s, 3H), 1.89 (d, J = 2.4 Hz, 6H), 1.75 (dd, J = 33.3, 12.4 Hz, 6H). 13C NMR (101 MHz, CD3OD): δ 157.4, 157.1, 157.0, 139.7, 130.2, 129.3, 127.6, 123.0, 116.0, 115.1, 103.2, 101.2, 51.5, 40.1, 38.0, 35.0, 29.0. HRMS (ESI): calcd for [M + H] + of C25H30NO3, 392.2226; found, 392.5237. trans-Resveratrol-3-O-β-D-glucopyranoside (5, Piceid). 2,3,4,6Tetra-O-acetyl-β-D-glucopyranosyl bromide was selected to be the glucosidation agent in the preparation of piceid. To a stirred 50 mL of acetonitrile were dissolved 228 mg of resveratrol (1 mmol) and 411 mg of the aforementioned glucosidation agent (1 mmol), and then 276 mg of Ag2CO3 (1 mmol) was added and stirred at ambient temperature for 8 h in dark. The reaction mixture was then filtered through a short Celite pad, the solvent was removed in vacuo, and the obtained residue was purified by silica gel column chromatography with petroleum ether/ethyl acetate mixture (v/v = 3:1) as eluent to afford the precursor of 5. The acetyl groups in glucose moiety were removed by reacting with NaOCH3. To 20 mL of methanolic solution of the precursor of 5 (140 mg, 0.25 mmol) was added 67.5 mg of NaOCH3 and stirred at room temperature for 6 h in the atmosphere of N2. The reaction mixture was acidified to pH = 2 and filtered. The liquid phase was concentrated in vacuo, and the residue was purified by thin layer chromatography with ethyl acetate/methanol mixture (v/v = 3:1) as eluent to give 5 as a white solid in a yield of 6% (25 mg). 1H NMR (400 MHz, DMSO-d6): δ 9.60 (s, 1H), 9.46 (s, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 16.3 Hz, 1H), 6.88 (d, J = 16.3 Hz, 1H), 6.77 (d, J = 8.4 Hz, 2H), 6.74 (s, 1H), 6.57 (s, 1H), 6.34 (s, C


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Figure 2. Modification of resveratrol by various structural features. at 37 °C for initiating the oxidation of DNA, and three of them were taken out at 30 min and cooled in ice water. Then 1.0 mL of PBS1 solution of 2-thiobarbituric acid (TBA) (1.00 g of TBA and 0.40 g of NaOH were dissolved in 100 mL of PBS1) and 1.0 mL of 3.0% aqueous solution of trichloroacetic acid were added and heated in boiling water for 30 min. After the test tubes were cooled in ice water, 1.5 mL of n-butanol was added and shaken vigorously to extract thiobarbituric acid reactive species (TBARS). The absorbance of organic layer was measured at 535 nm (Adetect) and compared with that in the control experiment (Aref). CuSO4 and glutathione (GSH) were dissolved in PBS1 to reach 75.0 and 90.0 mM as stock solutions, respectively. To 13.4 mL of PBS1 solution of DNA was added 1.0 mL of CuSO4 and 0.5 mL of GSH solution, and 0.1 mL of DMSO solution of resveratrol derivatives. The aforementioned mixture was aliquoted into test tubes, with each containing 2.0 mL (the final concentrations of DNA, Cu2+, GSH, and resveratrol were 2.00 mg/mL, 5.0, 3.0 mM, and 200 μM, respectively; the same volume of DMSO was contained in the control experiment to eliminate the influence from DMSO). The test tubes were incubated at 37 °C for initiating the oxidation of DNA, and three of them were taken out at 90 min and cooled in ice water. PBS1 solution of EDTA (1.0 mL, 30.0 mM) was added to chelate Cu2+, and then 1.0 mL of PBS1 solution of TBA and 1.0 mL of 3.0% aqueous solution of trichloroacetic acid were added and heated in boiling water for 30 min. After the test tubes were cooled in ice water, 1.5 mL of n-butanol was added and shaken vigorously to extract TBARS. The absorbance of organic layer was measured at 535 nm (Adetect) and compared with that in the control experiment (Aref). AAPH-Induced Oxidation of DNA. DNA was dissolved in phosphate buffered solution (PBS2: 8.1 mM Na2HPO4, 1.9 mM

NaH2PO4, 10.0 μM EDTA, pH = 7.4) to reach 2.24 mg/mL as the stock solution, and AAPH was also dissolved in PBS2 to reach 400 mM as the stock solution. To 13.4 mL of PBS2 solution of DNA was added 1.5 mL of PBS2 solution of AAPH and a certain volume of DMSO solution of resveratrol derivatives. The aforementioned mixture was aliquoted into test tubes, with each containing 2.0 mL (the final concentrations of DNA and AAPH were 2.00 mg/mL and 40.0 mM, respectively, and the final concentrations of resveratrol derivatives were shown in the corresponding chart in the Supporting Information; the same volume of DMSO was contained in the control experiment to eliminate the influence from DMSO). The test tubes were incubated at 37 °C for initiating the oxidation of DNA, and three of them were taken out every 2 h and cooled immediately in ice water. Then 1.0 mL of PBS2 solution of TBA (1.00 g of TBA and 0.40 g of NaOH dissolved in 100 mL of PBS2) and 1.0 mL of 3.0% aqueous solution of trichloroacetic acid were added, and the mixture was heated in boiling water for 15 min. After the test tubes were cooled in ice water, 1.5 mL of n-butanol was added and shaken vigorously to extract TBARS, whose absorbance was measured at 535 nm and plotted versus the reaction period. Statistical Analysis. All of the data were the average values from at least three independent measurements with the experimental error within 10%. The equations were analyzed by one-way ANOVA in Origin 7.0 professional Software, and p < 0.001 indicated a significance difference.

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RESULTS AND DISCUSSION Attaching Substituents to Position-2 of Resveratrol. The synthesis of resveratrol derivatives was mainly composed D


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Chemical Research in Toxicology of constructing CC by the Heck11 or Wittig reaction12 and of linking substituents with hydroxyl groups of resveratrol. We initially envisioned that introducing substituents into resveratrol directly was a convenient protocol for the preparation of resveratrol derivatives. Especially, attaching substituents to hydroxyl group-free positions in resveratrol might preserve biologically functional groups for resveratrol.26 A successful protocol as reported in literature27 was the formylation at position-2 via the Vilsmeier−Haack reaction; then −CHO acted as the linkage with other moieties. In contrast to the method reported in literature,27 as depicted in Figure 2, we herein performed the Vilsmeier−Haack reaction to afford 1 with phenolic hydroxyl groups in resveratrol not being protected by any groups (phenolic hydroxyl groups in resveratrol were methylated before the Vilsmeier−Haack reaction in the ref 27). We have found that introducing glucosyl or adamantyl group into an antioxidative molecule was able to enhance inhibitory effect against DNA oxidation.28 Thus, we attempted to bridge glucosamine or 1-adamantanamine with −CHO in 1 directly to simplify the synthetic operation, during which one drop of formic acid (as catalyst) and stirring at room temperature were applied to furnish imine between −CHO in 1 and amino group in glucosamine or 1-adamantanamine with mixed solution (THF/H2O) applied. As claimed in a previous report,29 “a synthetic nightmare” might unfortunately be met when resveratrol performed a reaction in the absence of the protection of hydroxyl groups, owing to the difficulty in the isolation of the target product. Thus, yields of 4 and 6 were just 17% and 6%, respectively, when thin-layer chromatography was used to isolate the products. Interestingly, the configuration of glucosyl group in 6 was transformed to be α-type although the configuration of D-glucosamine was β-type. A hydrolysis might perform on the glucosamine under acidic condition (one drop of formic acid as catalyst), while the configuration of hydroxyl group at position-1 in glucose moiety was changed via the recyclization. To avoid lowering yield in the case of 1 being the reagent, we had to use 1a as the starting material for preparing 6a, in which hydroxyl groups in glucosamine were protected by acetylation as well. Another benefit from the protection of hydroxyl groups in resveratrol by tert-butyldimethylsilyl chloride (TBDMSCl) and by acetylation in glucosamine was to dissolve all of the reagents in a homogeneous solution (methanol used herein). Then we attempted to reduce the CN in 6a to form amine (−CH2NH−) by NaBH4, and the acetyl groups at glucose moiety was removed under K2CO3, followed by the removal of TBDMS group under acidic condition. Figure 3 outlined that 1 H NMR peak of −CHN- in 6 (8.71 ppm) moved to 4.08 ppm, indicating that −CHN− in 6 was transformed to −CH2NH− in 7. For better comparison of the antioxidative effects of glucosyl imine or amine of resveratrol (6 or 7) with that of resveratrol glucoside (a glucosyl moiety attaching to one of hydroxyl group in resveratrol), we synthesized piceid (5, with 2,3,4,6tetra-O-acetyl-β-D-glucopyranosyl bromide being the glycosyl donor) as a reference compound. In contrast to the method reported in a literature,30 we employed resveratrol to react with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl bromide directly. However, the low yield of piceid (5) was due to the sensitivity toward ambient oxidation when the acetyl groups at glycose moiety in 5 were removed by CH3ONa. Moreover, it was necessary to test the influence of −CHO in 1 and −CH2OH in

Figure 3. Comparison on 1H NMR peak of imine in 6 and that of amine in 7.

2 on the antioxidative effect of resveratrol. The lipoic acid was a widely used antioxidant, and whether it could enhance antioxidative effect after forming a hybrid with resveratrol was concerned in this work. Thus, −CH2OH in 2a took place the esterification with lipoic acid, and compound 3 was given after the deprotection of TBDMS groups. Inhibitory Effects against •OH- and Cu2+/GSHInduced Oxidations of DNA. The ribose moieties in DNA were readily oxidized by hydroxyl radical (•OH), leading to the degradation of DNA.31 Chemically, a mixture of tetrachlorohydroquinone (TCHQ, 3.0 mM) and H2O2 (1.5 mM) was able to generate •OH, which was applied to mimic DNA (2.00 mg/mL) undergoing •OH-mediated oxidation. The products resulting from DNA oxidation could be colorized by thiobarbituric acid (TBA), and the absorbance of thiobarbituric acid reactive species (TBARS, λmax = 535 nm) resulting from the control experiment (performing 30 min) was assigned as 100%. The absorbances of TBARS in the presence of resveratrol derivatives (200 μM) were compared with that in the control experiment and expressed as TBARS percentages. As depicted in Figure 4, a low TBARS% meant that the corresponding resveratrol derivative possessed high inhibitory effect against •OH-induced oxidation of DNA. However, data in the upper panel of Figure 4 did not provide positive results because TBARS percentages limited from 83.9% to 97.2%, not different significantly. A recently published report suggested that resveratrol functioned as •OH-scavenger by all possible mechanisms, but mainly followed sequential electron proton transfer (SEPT) and radical adduct formation (RAF).26 This might be helpful for understanding the results obtained in the present study. The −CHO as an electron-withdrawing group in 1 was not beneficial for electrons being transferring from resveratrol skeleton to •OH (following SEPT mechanism), while position-2 was an active site for taking place radicaladdition by •OH (following RAF mechanism). In the presented work, position-2 was applied to link with other structural moieties and thus could not play antioxidative role by absorbing •OH at this position. However, compound 6, which consisted of a CN bond to enlarge the conjugation system of resveratrol, exhibited similar inhibitory effect (TBARS% = 84.8%) as resveratrol (TBARS% = 83.9%) in this experimental system. Subsequently, we tested the inhibitory effects of these resveratrol derivatives against DNA oxidation mediated by copper ion (Cu2+) and glutathione (GSH).32 A mixture of 5.0 mM Cu2+ and 3.0 mM GSH was used to produce glutathione radical (GS•) for oxidizing 2.00 mg/mL DNA at 37 °C. After DNA underwent Cu2+/GSH-mediated oxidation for 90 min, E


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substrate for a period, the inhibition period (tinh) was proved to correlate proportionally with the concentration of the antioxidant (see eq 1). In eq 1, Ri stood for the initiation rate of the radical-induced oxidation, and n referred to the stoichiometric factor, which meant the number of the radical-propagation terminated by one molecule of the antioxidant:36 t inh = (n/R i) [antioxidant]

(1)

We have measured the relationship between the concentration of resveratrol and the inhibition period (tinh) in the experimental system of AAPH-induced oxidation of DNA (see eq 2):37 t inh = 1.10 ( ±0.04) [resveratrol] + 38.62 (± 3.05)

(2)

It was safely assumed that the initiation rate (Ri) of DNA oxidation was equal to the radical-generation rate (Rg = (1.4 ± 0.2) × 10−6 [AAPH] s−1 at 37 °C), namely, Ri = Rg = 1.4 × 10−6 × 40 mM s−1 = 3.36 μM min−1 because DNA sodium salt and AAPH were dissolved in the same water phase.38 The coefficient in eq 2, 1.10, was equivalent to n/Ri in eq 1, and the stoichiometric factor (n) of resveratrol was the product of the coefficient in eq 2 multiplying Ri. Thus, the n of resveratrol was 3.70, indicating that one molecule of resveratrol was able to terminate 3.70 radical-propagations in the protection of DNA against AAPH-induced oxidation. We next attempted to measure inhibition periods (tinh) with various concentrations of resveratrol derivatives added (shown in Figure S8) and obtained correlations of tinh with concentrations of resveratrol derivatives (shown in Figure S9). Subsequently, equations of tinh ∼ [resveratrol derivative] were collected in Table S1, and stoichiometric factors (n) of resveratrol derivatives were calculated and outlined in Figure 5. It was found in Figure 5 that the n values of 4 and 5 were similar to that of resveratrol,

Figure 4. TBARS percentages in the DNA oxidation mediated by • OH (upper) and Cu2+/GSH (below) as well as structures of resveratrol derivatives.

TBARS percentages in the presence of resveratrol derivatives (200 μM) were measured and outlined in the below panel of Figure 4. A previous report revealed that the combination of Cu2+ with resveratrol could destroy DNA strand.33 We herein found that TBARS% of resveratrol was 94.1%, almost closing to the control experiment (100%). In addition, TBARS% in the case of additions of other resveratrol derivatives limited from 85.5% (7) to 94.3% (4), not different significantly with each other. As pointed out in the literature,33 DNA strand might be oxidized in the mixture of Cu2+ and resveratrol. In the presented study, we just appended a series of structural moieties to position-2 of resveratrol and did not vary the resveratrol skeleton. Hence, the resveratrol derivatives used herein might play the same role as resveratrol in the case of interacting with Cu2+ and functioned as resveratrol itself. Therefore, the inhibitory effects of the synthetic resveratrol derivatives approached to that of resveratrol in this experimental system. This encouraged us to find a quantitative index for characterizing the antioxidant effects of these compounds. Stoichiometric Factor (n) in AAPH-Induced Oxidation of DNA. Of interest to us was to quantitatively express inhibitory effects of these resveratrol derivatives against peroxyl radical-mediated oxidation of DNA. The guanine base in DNA was sensitive toward the oxidation by 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH, R-NN-R, R = −CMe2C(NH)NH2, ROO• resource),34 and oxidative products could also be detected via the reaction with TBA.35 Chemically, if an antioxidant was able to inhibit AAPH-induced oxidation of a

Figure 5. Stoichiometric factors (n) of resveratrol derivatives with antioxidative activities being cataloged as low (1, 2, and 3), middle (4 and 5), and high (6 and 7) groups. F


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Figure 6. Influence of structural factors on stabilities of radicals deriving from resveratrol derivatives.

while the n values of 1, 2, and 3 were lower than that of resveratrol. Exceptionally, the n values of 6 and 7 were higher than that of resveratrol, revealing that only 6 and 7 exhibited higher antioxidative effects than resveratrol in this case. It was reported that installation of substituent to hydroxyl group was not beneficial for enhancing the antioxidative effect of resveratrol.26 The stoichiometric factor (n) of 5 was only 3.49, lower than that of 6 and 7 (4.74 and 4.97, respectively), and even lower than that of resveratrol, 3.70. Thus, the glucoside of resveratrol did not show higher antioxidative effect than resveratrol. This fact reminded us to equip substituents with hydroxyl group-free positions in resveratrol. However, as depicted in Figure 6, if a phenoxyl radical was produced at position-5, the single electron could be transferred via a series of resonance structures (from 8 to 10) to position-2, encountering the electron-withdrawing group, −CHO, which was not beneficial for stabilizing radical. Meanwhile, the intramolecular hydrogen bond produced between −CHO and adjacent −OH increased the bond dissociation enthalpy (BDE) of O−H to 412.0 kJ mol−1, 59.0 kJ mol−1 (ΔBDE) higher than that in resveratrol. The −CHO even increased ΔBDE of O−H of 23.1 and 8.9 kJ mol−1 when −OH located at 5- and 4′-positions, respectively.39 Apparently, introducing −CHO into position-2 made H atoms in all of the −OH not to be abstracted readily. After −CHO in 1 was reduced to form −CH2OH, stoichiometric factors (n) of 2 decreased to 1.99, the lowest value among these resveratrol derivatives. The intramolecular hydrogen bond between the oxygen atom in −CH2OH and the hydrogen atom in the adjacent phenolic −OH still hindered the hydrogen atom in phenolic −OH to be transferred to radicals. As depicted in Figure S10, lipoic acid just decreased the oxidative rate of AAPH-induced oxidation of DNA and cannot result in tinh. The stoichiometric factor (n) of 3 was 2.04, lower than that of resveratrol. In addition, we have found that introducing adamantanyl group into antioxidative molecule was able to ameliorate inhibitory effect against AAPH-induced oxidation of DNA,28 but herein, the combination of

adamantanyl group with resveratrol just recovered stoichiometric factor (n) of 4 to 3.55, still lower than that of resveratrol (3.70). The aforementioned results implied that both lipoic and adamantanyl groups could not bring resveratrol with an ability to affiliate DNA strand. On the other hand, compound 6, which was composed of a glucose moiety as an imine, increased the n value to 4.74, higher than that of resveratrol (3.70). The n value of 7 was further increased to 4.97; therefore, attaching glucose moiety to hydroxyl group-free position was able to enhance the inhibitory effect of resveratrol against DNA oxidation. A detail in the structures of 6 and 7 aroused our interests, and we tried to use conformation analysis to give an explanation. In compound 6, −OH at position-1 of glucosyl moiety located at axial bond, while −OH at position-1 of glucosyl moiety in compound 7 might locate at axial or equatorial bond. As shown in Figure 6, the axial −OH in 6 might form a hydrogen bond with N atom in imine. The same type of hydrogen bond could also be produced in compound 7. Moreover, the amine group in 7 might form a hydrogen bond with the −OH in resveratrol moiety. Thus, acidic and basic conditions applied in the deprotection of hydroxyl groups (see Figure 2) might change the configuration of −OH at position1 in glucosyl group.

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CONCLUSIONS Resveratrol derivatives endowed with unique antioxidative properties. We herein provided with a protocol for the synthesis of resveratrol derivatives via the formylation at position-2 of resveratrol. Although both −CHO and −CH2OH at position-2 of resveratrol did not exert positive effects against DNA oxidation, and attaching lipoic acid and adamantanamine to position-2 even showed negative effects, bridging glucose moiety with −CHO at position-2 as an imine or amine ameliorated the antioxidative property, and the inhibitory effect against DNA oxidation was much higher than that of resveratrol glucoside. Therefore, preserving hydroxyl groups and attaching other modified moieties at position-2 might G


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(12) Nakao, S., Mabuchi, M., Wang, S., Kogure, Y., Shimizu, T., Noguchi, K., Tanaka, A., and Dai, Y. (2017) Synthesis of resveratrol derivatives as new analgesic drugs through desensitization of the TRPA1 receptor. Bioorg. Med. Chem. Lett. 27, 3167−3172. (13) Goh, Y. L., Cui, Y. T., Pendharkar, V., and Adsool, V. A. (2017) Toward resolving the resveratrol conundrum: Synthesis and in vivo pharmacokinetic evaluation of BCP-resveratrol. ACS Med. Chem. Lett. 8, 516−520. (14) Xu, P., Zhang, M., Sheng, R., and Ma, Y. (2017) Synthesis and biological evaluation of deferiprone-resveratrol hybrids as antioxidants, Aβ1−42 aggregation inhibitors and metal-chelating agents for Alzheimer’s disease. Eur. J. Med. Chem. 127, 174−186. (15) Tang, J.-J., Fan, G.-J., Dai, F., Ding, D.-J., Wang, Q., Lu, D.-L., Li, R.-R., Li, X.-Z., Hu, L.-M., Jin, X.-L., and Zhou, B. (2011) Finding more active antioxidants and cancer chemoprevention agents by elongating the conjugated links of resveratrol. Free Radical Biol. Med. 50, 1447−1457. (16) Poutiainen, P. K., Palvimo, J. J., Hinkkanen, A. E., Valkonen, A., Väisänen, T. K., Laatikainen, R., and Pulkkinen, J. T. (2013) Discovery of 5-benzyl-3-phenyl-4,5-dihydroisoxazoles and 5-benzyl-3phenyl-1,4,2-dioxazoles as potent firefly luciferase inhibitors. J. Med. Chem. 56, 1064−1073. (17) Domazetovic, V., Fontani, F., Tanini, D., D’Esopo, V., Viglianisi, C., Marcucci, G., Panzella, L., Napolitano, A., Brandi, M. L., Capperucci, A., Menichetti, S., Vincenzini, M. T., and Iantomasi, T. (2017) Protective role of benzoselenophene derivatives of resveratrol on the induced oxidative stress in intestinal myofibroblasts and osteocytes. Chem.-Biol. Interact. 275, 13−21. (18) Zhu, Y., Fu, J., Shurlknight, K. L., Soroka, D. N., Hu, Y., Chen, X., and Sang, S. (2015) Novel resveratrol-based aspirin prodrugs: Synthesis, metabolism, and anticancer activity. J. Med. Chem. 58, 6494−6506. (19) Li, S., Zhang, W., Yang, Y., Ma, T., Guo, J., Wang, S., Yu, W., and Kong, L. (2016) Discovery of oral-available resveratrol-caffeic acid based hybrids inhibiting acetylated and phosphorylated STAT3 protein. Eur. J. Med. Chem. 124, 1006−1018. (20) Lu, C., Guo, Y., Yan, J., Luo, Z., Luo, H.-B., Yan, M., Huang, L., and Li, X. (2013) Design, synthesis, and evaluation of multitargetdirected resveratrol derivatives for the treatment of Alzheimer’s disease. J. Med. Chem. 56, 5843−5859. (21) Jeřab́ ek, J., Uliassi, E., Guidotti, L., Korábečný, J., Soukup, O., Sepsova, V., Hrabinova, M., Kuča, K., Bartolini, M., Peña-Altamira, L. E., Petralla, S., Monti, B., Roberti, M., and Bolognesi, M. L. (2017) Tacrine-resveratrol fused hybrids as multi-target-directed ligands against Alzheimer’s disease. Eur. J. Med. Chem. 127, 250−262. (22) Cardullo, N., Spatafora, C., Musso, N., Barresi, V., Condorelli, D., and Tringali, C. (2015) Resveratrol-related polymethoxystilbene glycosides: Synthesis, antiproliferative activity, and glycosidase inhibition. J. Nat. Prod. 78, 2675−2683. (23) Lu, D.-L., Ding, D.-J., Yan, W.-J., Li, R.-R., Dai, F., Wang, Q., Yu, S.-S., Li, Y., Jin, X.-L., and Zhou, B. (2013) Influence of glucuronidation and reduction modifications of resveratrol on its biological activities. ChemBioChem 14, 1094−1104. (24) Xie, K., Chen, R., Li, J., Wang, R., Chen, D., Dou, X., and Dai, J. (2014) Exploring the catalytic promiscuity of a new glycosyltransferase from Carthamus tinctorius. Org. Lett. 16, 4874−4877. (25) Jackson, S. P., and Bartek, J. (2009) The DNA-damage response in human biology and disease. Nature 461, 1071−1078. (26) Iuga, C., Alvarez-Idaboy, J. R., and Russo, N. (2012) Antioxidant activity of trans-resveratrol toward hydroxyl and hydroperoxyl radicals: A quantum chemical and computational kinetics study. J. Org. Chem. 77, 3868−3877. (27) Ruan, B.-F., Cheng, H.-J., Ren, J., Li, H.-L., Guo, L.-L., Zhang, X.-X., and Liao, C. (2015) Novel 2H-chromen-2-one derivatives of resveratrol: Design, synthesis, modeling and use as human monoamine oxidase inhibitors. Eur. J. Med. Chem. 103, 185−190. (28) Zhao, P.-F., and Liu, Z.-Q. (2017) 2-Isocyano glucose used in Ugi four-component reaction: An approach to enhance inhibitory effect against DNA oxidation. Eur. J. Med. Chem. 135, 458−466.

become a promising protocol for ameliorating antioxidative effect of resveratrol.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.8b00136.

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NMR and HRMS spectra of resveratrol derivatives; inhibitory effects of resveratrol derivatives against AAPH-induced oxidation of DNA (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Zai-Qun Liu: 0000-0001-9573-0724 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Jilin Provincial Science and Technology Department, China, is acknowledged gratefully (20160101317JC).

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REFERENCES

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Hybrid of Resveratrol and Glucosamine: An Approach To Enhance

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