The design,synthesis,antioxidant,and antihypoxia activities of two new hydroxydaidzein derivatives

Synthesis of 8,3’,5’-tribromo-7,4’-dihydroxyisoflavone (1)

To a stirring solution of daidzein (20 mmol, 5.08 g) in CH₂Cl₂ (200 mL) and ethanol (20 mL) was added dropwise Br₂ (4.1 mL). The reaction mixture was stirred at room temperature (RT, 25 °C) for 2.5 h, followed by treatment with saturated NaHSO₃ solution (5 mL) to remove any remaining traces of Br₂. The organic solvent was removed under reduced pressure to give a precipitate, which was collected by filtration, washed with distilled water, dried under vacuum, and washed with hot ethyl acetate to afford compound 1 as white powdery solid (yield 9.2 g, 92%); m.p. 253–254 °C. IR (KBr) v_max: 3450, 3074, 2927, 1619, 1594, 1434, 1285, 1238, 1216, 1118, 1076, 783 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ 11.70 (s, 1H), 10.11 (s, 1H), 8.62 (s, 1H, H-2), 7.97 (d, J = 8.8 Hz, 1H, H-5), 7.80 (s, 2H, H-2’-H, H-6’), and 7.14 (d, J = 8.8 Hz, 1H, H-5). The ¹H NMR data are in good agreement with the literature. ¹⁹ ESI-MS: m/z = 488.7 [M + H]⁺.

Synthesis of 8,3’,5’-tribromo-7,4’-dimethoxyisoflavone (2)

To a stirred solution of compound 1 (4.90 g, 10 mmol) in dry acetone (150 mL) were added anhydrous K₂CO₃ (2.76 g, 20 mmol) and dimethyl sulfate (2.0 mL, 20 mmol). The reaction mixture was heated at reflux and stirred for 6 h. The resulting mixture was cooled to RT, quenched with ammonium hydroxide (2 mL), and then diluted with water (150 mL). The acetone was removed under reduced pressure. The crude residue was filtered, washed with water, and dried under vacuum to afford compound 2 (yield 5.06 g, 98%) as a white powdery solid; m.p. 295–296 °C. IR (KBr) v_max: 2981, 2946, 1655, 1616, 1597, 1424, 1284, 1273, 1217, 1082, 1051, 784 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 8.26 (d, J = 8.8 Hz, 1H, H-5), 8.06 (s, 1H, H-2), 7.74 (s, 2H, H-2’-H, H-6’), 7.08 (d, J = 9.2 Hz, 1H, H-6), 4.06 (s, 3H, –OCH₃), 3.93 (s, 3H, –OCH₃). ESI-HRMS: m/z = [M + H]⁺ calcd for C₁₇H₁₁Br₃O₄: 518.8270; found: 518.8260.

Synthesis of 7,8,3’,4’,5’-pentamethoxyisoflavone (3)

A solution of 25% sodium methoxide in methanol (70 mL) was added to a suspension of CuBr (2.14 g, 15 mmol) in DMF (5 mL). The mixture was stirred at RT for 1 h and added to a solution of 2 (2.08 g, 4 mmol) in DMF (5 mL) at 120 °C. After stirring for 3 h, the reaction mixture was cooled to RT, poured into ice water, adjusted to pH 6 using 2 M HCl (aq), and extracted with ethyl acetate (80 mL × 3). The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (petroleum ether/ethyl acetate, v/v, 2/1) to give compound 3 (yield 1.01 g, 68%) as white solid needles; m.p. 142–143 °C (144 °C). ²⁰ IR (KBr) v_max: 1649, 1619, 1584, 1511, 1454, 1433, 1320, 1290, 1245, 1131, 1081, 818 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 8.06 (d, J = 9.2 Hz, 1H, H-5), 8.05 (s, 1H, H-2), 7.10 (d, J = 8.8 Hz, 1H, H-6), 6.79 (s, 2H, H-2’, H-6’), 4.02 (s, 3H, –OCH₃), 4.01 (s, 3H, –OCH₃), 3.91 (s, 6H, –OCH₃), 3.89 (s, 3H, –OCH₃). ¹³C NMR (100 MHz, CDCl₃): δ 175.9 (C-4), 156.5 (C-7), 153.2 (C-2), 152.7 (C-3’, C-5’), 150.5 (C-9), 138.1 (C-4’), 136.6 (C-8), 127.3 (C-1’), 124.8 (C-3), 121.8 (C-5), 119.2 (C-10), 110.2 (C-6), 106.3 (C-2’, C-6’), 61.7 (−OCH₃), 60.9 (–OCH₃), 56.5 (–OCH₃), 56.2 (2C, –OCH₃). ESI-HRMS: m/z = [M + H]⁺ calcd for C₂₀H₂₀O₇: 373.1287; found: 373.1282.

Synthesis of 7,8,3’,4’,5’-pentahydroxyisoflavone (8,3’,5’-THD)

To a stirred solution of AlCl₃ (998 mg, 7.5 mmol) in CH₂Cl₂ (15 mL) was added dropwise dimethyl sulfide (548 µL) at 5 °C, and then compound 3 (187 mg, 0.5 mmol) was added and the reaction mixture was stirred at 25 °C for 10 h. Upon cooling to 0 °C, 10% HCl (30 mL) was added, and the resulting mixture was stirred for an additional 2 h at RT. The organic solvent was removed under vacuum to form a precipitate, which was collected by filtration and recrystallized from aqueous methanol to give 8,3’,5’-THD (yield 137 mg, 91%) as a light yellow solid; m.p. >320 °C. IR (KBr) v_max: 3385, 1627, 1604, 1474, 1449, 1407, 1325, 1181, 1023, 779 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ 10.27 (s, C-OH), 9.40 (s, C-OH), 8.86 (s, C-OH), 8.25 (s, 1H, H-2), 7.46 (1H, d, J = 8.8 Hz, H-5), 6.96 (1H, d, J = 8.8 Hz, H-6), 6.51 (2H, s, H-2’, H-6’). ¹³C NMR (100 MHz, DMSO-d₆): δ 175.5 (C-4), 153.0 (C-7), 150.3 (C-2), 147.0 (C-9), 146.1 (C-3’, C-5’), 133.5 (C-4’), 133.3 (C-8), 123.8 (C-1’), 122.6 (C-5), 118.0 (C-3), 116.1 (C-6), 114.5 (C-10), 108.5 (C-2’, C-6’). ESI-HRMS: m/z = [M + H]⁺ calcd for C₁₅H₁₀O₇: 303.0504; found: 303.0499.

Synthesis of 6,8,3’,5’-tetrabromo-7,4’-dihydroxyisoflavone (4)

To a stirring solution of daidzein (10 mmol, 2.50 g) in ethanol (150 mL) was added dropwise Br₂ (3.0 mL) at RT. The resulting mixture was then heated at reflux and stirred for 1 h. Water (200 mL) was added to the resulting mixture under boiling conditions. After cooling to RT, the obtained precipitate was collected by filtration, washed with water, and dried under vacuum to give compound 4 (yield 5.5 g, 96%) as a white powdery solid; m.p. >320 °C. IR (KBr) v_max: 3439, 3085, 1637, 1615, 1593, 1432, 1293, 1283, 1179, 1075, 855 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ 10.14 (s, H, C-OH), 8.66 (s, 1H, H-5), 8.18 (s, 1H, H-2), 7.80 (s, 2H, H-2’, H-6’). ¹³C NMR (100 MHz, DMSO-d₆): δ 173.6 (C-4), 156.5 (C-7), 155.3 (C-4’), 153.4 (C-9), 151.1 (C-2), 132.9 (C-5), 128.5 (C-2’, C-6’), 126.0 (C-1’), 121.4 (C-3), 118.8 (C-10), 112.0 (C-3’, C-5’), 110.4 (C-8), 100.2 (C-6). ESI-HRMS: m/z = [M + H]⁺ calcd for C₁₅H₆Br₄O₄: 570.7041; found: 570.7031.

Synthesis of 6,8,3’,5’-tetrabromo-7,4’-dimethoxyisoflavone (5)

To a stirred solution of compound 4 (2.85 g, 5 mmol) in dry acetone (100 mL) was added anhydrous K₂CO₃ (2.07 g, 15 mmol) and dimethyl sulfate (1.5 mL, 15 mmol). The reaction mixture was heated to reflux and stirred for 6 h. The resulting mixture was cooled to RT, quenched with ammonium hydroxide solution (1.5 mL), and then diluted with water (100 mL). The acetone was removed under reduced pressure. The crude residue was filtered, washed with distilled water, and dried under vacuum to give compound 5 (2.84 g, 95%) as a white powdery solid; m.p. 209–210 °C. IR (KBr) v_max: 2944, 2830, 1653, 1615, 1543, 1471, 1428, 1408, 1268, 1232, 870, 846, 741, 659 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 8.46 (s, 1H, H-2), 8.10 (s, 1H, H-5), 7.71 (s, 2H, H-2’, H-6’), 4.02 (s, 3H, –OCH₃), 3.92 (s, 3H, –OCH₃). ¹³C NMR (100 MHz, CDCl₃): δ 173.7 (C-4), 159.0 (C-7), 154.4 (C-4’), 153.5 (C-9), 153.2 (C-2), 132.9 (C-5), 129.6 (C2’, C-6’), 129.4 (C-1’), 122.9 (C-3), 122.4 (C-10), 118.3 (C-6), 115.9 (C-3’, C-5’), 108.0 (C-8), 61.2 (–OCH₃), 60.7 (–OCH₃). ESI-HRMS: m/z = [M + H]⁺ calcd for C₁₇H₁₀Br₄O₄: 598.7354; found: 598.7344.

Synthesis of 6,7,8,3’,4’,5’-hexamethoxyisoflavone (6)

A solution of 25% sodium methoxide in methanol (50 mL) was added to the suspension of CuBr (1.43 g, 10 mmol) in DMF (4 mL). The mixture was stirred at RT for 1 h and then added to a solution of 6 (1.19 g, 2 mmol) in DMF (3 mL) at 120 °C. After stirring for 4 h, the reaction mixture was poured into ice water, adjusted to pH 6 using 2 M HCl (aq), and extracted with ethyl acetate (50 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (petroleum ether/ethyl acetate, v/v, 1/1) to give 7 (563 mg, 70%) as white solid needles; m.p. 164–165 °C (172 °C). ²⁰ IR (KBr) v_max: 1634, 1585, 1467, 1423, 1320, 1247, 1127, 845 cm^–1. ¹H NMR (400 MHz, CDCl₃): δ 8.08 (s, 1H, H-2), 7.47 (s, 1H, H-5), 6.81 (s, 2H, H-2’, H-6’), 4.06 (s, 3H, −OCH₃), 4.05 (s, 3H, –OCH₃), 3.97 (s, 3H, –OCH₃), 3.91 (s, 6H, –OCH₃), 3.89 (s, 3H, –OCH₃). ¹³C NMR (100 MHz, CDCl₃): δ 175.5 (C-4), 153.2 (C-2), 152.6 (C-3’, C-5’), 151.4 (C-6), 147.3 (C-7), 145.8 (C-9), 141.7 (C-8), 138.1 (C-4’), 127.4 (C-1’), 124.5 (C-3), 120.3 (C-10), 106.2 (C-2’, C-6’), 100.3 (C-5), 62.0 (–OCH₃), 61.5 (–OCH₃), 60.9 (–OCH₃), 56.3 (–OCH₃), 56.2 (2C, –OCH₃). ESI-HRMS: m/z = [M + H]⁺ calcd for C₂₁H₂₂O₈:403.1387; found: 403.1393.

Synthesis of 6,7,8,3’,4’,5’-hexahydroxyisoflavone (6,8,3’,5’-FHD)

Dimethyl sulfide (700 µL) was slowly added dropwise to a stirred solution of AlCl₃ (1.33 g, 10 mmol) in CH₂Cl₂ (15 mL) at 5 °C. Compound 6 (242 mg, 0.6 mmol) was then added, and the reaction mixture was stirred at RT for 12 h. After cooling to 0 °C, 10% HCl (30 mL) was added, and the resulting mixture was stirred for an additional 2 h at RT. The organic solvent was removed under vacuum to give a precipitate, which was collected by filtration, washed with distilled water, and recrystallized from aqueous methanol to afford 6,8,3’,5’-FHD (188 mg, 90%) as white solid needles; m.p. >320 °C. IR (KBr) v_max: 3437, 3230, 1607, 1540, 1485, 1372, 1335, 1297, 1033, 843 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ 8.22 (s, 1H, H-2), 7.00 (s, 1H, H-5), 6.53 (s, 2H, H-2’, H-6’). ¹³C NMR (100 MHz, DMSO-d₆): δ 175.1 (C-4), 152.6 (C-2), 146.1 (C-3’, C-5’), 144.9 (C-9), 141.8 (C-7), 140.1 (C-6), 134.1 (C-8), 133.4 (C-4’), 123.2 (C-1’), 123.0 (C-3), 116.7 (C-10), 108.5 (C-2’, C-6’), 99.0 (C-5). ESI-HRMS: m/z = [M + H]⁺ calcd for C₁₅H₁₀O₈: 319.0453; found: 319.0448.

DPPH radical scavenging assay

The DPPH radical scavenging assay was performed according to the reported method with slight modifications. ²¹ In brief, 100 μL of the sample in DMSO at various concentrations (0.0625–2.0 mmol L⁻¹) and 100 μL of DPPH solution (100 µM) in methanol were added to a 96-well plate and shaken. After incubation in the dark for 30 min at RT, the absorbance of the resulting solution was recorded at 517 nm using a microplate reader (SpectraMax i3, Molecular Devices, San Jose, CA, USA). The DPPH free-radical scavenging rate was calculated using the equation

DPPH scavenging activity ( % ) = ( 1 − ( A s − A b ) A c ) × 100 %

(1)

where A_b is the absorbance of the blank control (containing all reagents except samples), A_c is the absorbance of the sample control (containing all reagents except DPPH), and A_s is the absorbance of the sample (containing all reagents with the samples). The antioxidant activity is expressed as the value IC₅₀ value (mmol L⁻¹), which is calculated as the concentration of sample required to scavenge 50% of the DPPH radicals. A low IC₅₀ value corresponded with higher antioxidant activity.

Superoxide anion (O₂^−•) radical scavenging assay

The O₂^−• scavenging activity was performed using the phenazine methosulfate (PMS)–nicotinamide adenine dinucleotide (NADH)–nitrotetrazolium blue chloride (NBT) system with slight modifications. ²² In brief, 100 µL of the sample in DMSO at various concentrations (0.015625–1.0 mmol mL⁻¹), 50 µL of NBT solution (0.2 mM) in distilled water, and 50 µL of NADH solution (0.5 mmol L⁻¹) in 0.1 M Tris–HCl, pH 8.0 were added to a 96-well plate. PMS solution (50 µL, 25 µM) in distilled water was added to the mixture, which was shaken and then incubated for 10 min at RT. The absorbance of the resulting solution at 570 nm was recorded using a microplate reader (SpectraMax i3, Molecular Devices). The superoxide scavenging rate was calculated using equation (1), where A_c is the absorbance of the sample control (containing all reagents except PMS), and the other parameters are the same as mentioned above.

NO radical scavenging assay

The NO radical scavenging assay was measured by the Griess reaction with some modifications. ²³ In brief, 50 µL of the sample in DMSO at various concentrations (0.0625–2.0 mmol mL⁻¹) and 50 µL of sodium nitroprusside (20 mmol L⁻¹ in phosphate buffer, pH 7.4) were added to a 96-well plate and shaken. After incubation under light for 1.5 h at RT, 50 µL of 0.33% (w/v) sulfanilamide (in 20% glacial acetic acid) and 50 µL of 0.1% (w/v) naphthyl ethylenediamine hydrochloride were added to the resulting solution, which was further incubated for 30 min at RT. The absorbance was recorded at 540 nm in a microplate reader (SpectraMax i3, Molecular Devices). The NO radical scavenging rate was calculated according to equation (1), where A_c is the absorbance of the sample control (containing all reagents except sodium nitroprusside), and the other parameters are the same as mentioned above.

Reducing power assay

The reducing power assay was performed according to a previous reported method with slight modification. ²⁴ In brief, 100 μL of the sample in DMSO at various concentrations (0.03125–1.0 mmol L⁻¹) were mixed with 2.5 mL of 0.2 mol L⁻¹ sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% (w/v) potassium ferricyanide and incubated for 30 min at 50 °C. After cooling to RT, 2.5 mL of 10% trichloroacetic acid (TCA) was added to the mixture, which was then centrifuged at 3000 r/min for 10 min. The upper layer fraction (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride and reacted at RT for 30 min. The absorbance was recorded at 700 nm using a UV-Vis spectrophotometer (NanoPhotometer-NP80, Implen, Westlake Village, CA, USA). A higher absorbance indicated a stronger reducing power.

Phosphomolybdenum assay

The TAC of the sample was determined using the phosphomolybdate method. ²⁵ In brief, 100 µL of the sample in DMSO at various concentrations (0.03125–1.0 mmol L⁻¹) was mixed with 1.0 mL of the reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). After incubation at 95 °C for 90 min and then rapid cooling to RT, the absorbance of the resulting mixture was recorded at 695 nm using UV-Vis spectrophotometry (NanoPhotometer-NP80, Implen). A higher absorbance indicates a higher total antioxidant activity. Five assays were performed using Vitamin (V_C) as the positive control. All samples were analyzed in triplicate.

Cell culture and treatment

Rat pheochromocytoma (PC12) cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). As a normoxic control, cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Bioss, Beijing, China) supplemented with 10% fetal bovine serum (FBS) (Bioss) and 1% streptomycin–penicillin (Bioss) in a humidified incubator with 95% air and 5% CO₂ at 37 °C. To simulate hypoxia injury, the cells were incubated for 24 h at 37 °C in a hypoxic incubator containing 1% O₂, 94% N₂, and 5% CO₂.

Cell viability assay

Cell survival assays were carried out using the cell counting kit-8 (CCK-8) according to the manufacturer’s instructions. Samples were dissolved in DMSO and subsequently diluted in DMEM with the final concentration of DMSO less than 0.1% (v/v). PC12 cells were cultured in a 96-well plate for 24 h and pre-treated with or without samples at a concentration of 1 μmol L⁻¹ for 1 h. Cells were then cultured in normoxic or hypoxic conditions for 24 h. The medium was discarded and then 100 µL of fresh medium containing 10 µL of CCK-8 solution (Bioss, Beijing, China) was added to each well. After incubation for 2 h, the absorbance at 450 nm was obtained using a microplate reader (SpectraMax i3, Molecular Devices). The relative cell viability was calculated as the percentage of normoxic control.