General Melting points (m.p.) were determined on a SGWX-4 micro-melting point apparatus and are uncorrected. NMR spectra were recorded on Varian Mercury-300 spectrometer (300 MHz for 1H and 75 MHz for 13C) or Varian Mercury-400 spectrometer (400 MHz for 1H and 100 MHz for 13C), chemical shifts of 1H and 13C spectra were recorded with tetramethylsilane as internal standard (CDC13 δH 7.26, δC 77.2), and coupling constants were reported in hertz. Mass spectra were obtained on a ZAB-2F or JEOLDX-300 spectrometer. Optical rotations were measured on WZZ-3 polarimeter calibrated at the sodium Dline (598 nm). Reactions where exclusion of water was necessary were performed according to Ref. [25]. TLC was carried out on silica gel (GF254) under UV light. Column chromatography was run on silica gel (200–300 mesh) or alumina from Qingdao Ocean Chemical Factory.
Shikonin (3)
Shikonin was extracted from Lithospermum erythrorhizon according to the procedure described by Birch [26]. Red-brownish needles, m.p. 145–146 °C (from CH3OH) (lit. m.p. 146–147 °C [27]); [α]
25D
+ 126.5° (c 0.2, C6H6), (lit. +138° [2]).
(R)-5,8-dihydroxyl-2-(5,5-dimethyl-2-tetrahydrofuranyl)-1,4-naphthaquinone, (+) cycloshikonin (4)
Cycloshikonin was prepared from shikonin by the method proposed previously [2]. Yield: 98%. Solid, m.p. 78–80 °C (from CH3OH) (lit. m.p. 79–80 °C [2]); [α]
25D
+ 156.6° (c 0.33, CHCl3).1H NMR (300 MHz, CDCl3) δ: 12.53 (s, 1H, ArOH), 12.52 (s, 1H, ArOH), 7.23–7.19 (m, 3H, ArH, QuinoneH), 5.17 (dd, 1H, J = 6.3, 5.7 Hz, CH), 2.66–2.62 (m, 1H, CH
2
), 1.93–1.91 (m, 1H, CH
2
), 1.90–1.89 (m, 1H, CH
2
), 1.88–1.74 (m, 1H, CH
2
), 1.38 (s, 3H, CH
3), 1.35 (s, 3H, CH
3). 13C NMR (75 MHz, CDCl3) δ: 182.5, 181.5, 164.2, 163.7, 133.1, 132.0, 131.5, 131.4, 112.3, 111.9, 82.3, 74.7, 38.9, 33.7, 28.9, 28.0. MS (EI, m/z): 288 [M+], 255, 232, 219.
(R)-2-(5,5-dimethyl-2-tetrahydrofuranyl)-1,4,5,8-tetramethoxynaphthalene (5)
To a solution of 4 (5 g, 17.3 mmol) and tetrabutylammonium bromide (1.0 g) in THF (160 mL) and water (80 mL) was added sodium dithionite (15.1 g, 86.3 mmol). After stirring for 15 min, NaOH (13.9 g, 0.35 mol) was added at room temperature. Dimethyl sulfate (21 mL) was added dropwise in 10 min, and the mixture was refluxing for 24 h. The product was separated by partitioning between water and DCM. The crude product was purified by column chromatography over silica gel with ethyl acetate/petroleum ether (1/4, v/v) to give 5.46 g of pale-yellow oil. Yield: 91%. [α]
25D
+139.2o (c 0.2, CHCl3); 1H NMR (300 MHz, CDCl3) δ: 7.12 (s, 1H, ArH), 6.80 (s, 2H, ArH), 5.52 (m, 1H, CH), 3.99 (s, 3H, OCH
3), 3.95 (s, 3H, OCH
3), 3.93 (s, 3H, OCH
3), 3.75 (s, 3H, OCH
3), 2.54–2.48 (m, 2H, CH
2
), 1.94–1.84 (m, 2H, CH
2
), 1.45 (s, 3H, CH
3), 1.40 (s, 3H, CH
3).13C NMR (75 MHz, CDCl3) δ: 152.7, 150.8, 149.6, 145.8, 133.2, 122.0, 119.5, 107.5, 106.9, 105.4, 80.4, 74.5, 61.7, 51.2, 56.3, 56.2, 38.5, 34.4, 28.3, 27.6. MS (ESI, %): 369 (M+Na+, 100), 401 (M++NaOCH3, 45) and no parent peak was observed. HRMS (ESI) calcd. for C20H27O5
+: 347.1853 [M+H]+; found: 347.1856.
(R)-2-(1,4-diacetoxyl-4-methylpentyl)-1,4,5,8-tetramethoxynaphthalene (6) and 2-(4-acetoxyl-4-methyl-2-pentenyl)-1,4,5,8-tetramethoxynaphthalene (15)
A mixture of 5 (2 g, 5.8 mmo1) and p-toluenesulfonic acid monohydrate (1.14 g, 6 mmol) in acetic anhydride was allowed to stir overnight at −16 °C, and then the reaction mixture was diluted with methanol to quench excessive acetic anhydride and extracted with ethyl acetate. After the usual work-up, the residue was purified by column chromatography over silica gel with ethyl acetate/petroleum ether (1/3, v/v) as an eluent to give 2.28 g of pale-yellow oil. Yield: 88%. [α]
25D
+142.2° (c 0.2, CHCl3). 1H NMR (300 MHz, CDCl3) δ: 6.85 (s, 1H, ArH), 6.83 (s, 2H, ArH), 6.32 (t, 1H, J = 7.8 Hz, CH), 3.94 (s, 3H, OCH
3), 3.90 (s, 3H, OCH
3), 3.88 (s, 3H, OCH
3), 3.84 (s, 3H, OCH
3), 2.12 (s, 3H, OCOCH
3), 1.93–1.71 (m, 5H, CH
2, OCOCH
3), 1.41 (s, 3H, CH
3), 1.39 (s, 3H, CH
3). 13C NMR (75 MHz, CDCl3) δ: 170.5, 170.4, 153.8, 151.6, 150.7, 147.1, 130.9, 122.9, 121.1, 109.2, 108.1, 105.1, 81.9, 71.1, 62.7, 58.2, 57.7, 57.1, 37.1, 30.8, 26.2, 26.0, 22.6, 21.5. MS (ESI, %): 471 (M+Na+, 100), 503 (M++NaOCH3, 31) and no parent peak was observed. HRMS (ESI) calcd. for C24H33O8
+: 449.2170 [M+H]+, found: 449.2166.
The same operation as compound 6 was done at room temperature, major by-product 15 could be obtained as pale-yellow oil. 1H NMR (300 MHz, CDCl3) δ: 6.99 (s, 1H, ArH), 6.90 (d, 1H, J = 15.6 Hz, CH=CH), 6.83 (s, 2H, ArH), 6.28 (m, 1H, CH=CH), 4.00 (s, 3H, OCH
3), 3.95 (s, 3H, OCH
3), 3.84 (s, 3H, OCH
3), 3.73 (s, 3H, OCH
3), 2.78 (d, 2H, J = 6.6 Hz, CH
2), 2.02 (s, 3H, OCOCH
3), 1.52 (s, 6H, CH
3). 13C NMR (75 MHz, CDCl3) δ: 171.2, 153.6, 151.3, 150.5, 147.2, 131.0, 122.2, 119.1, 109.5, 105.8, 105.3, 81.8, 71.0, 62.4, 58.0, 57.5, 57.3, 37.0, 30.6, 26.3, 26.1, 22.7. MS (ESI, %): 411 (M+Na+, 100), 443 (M++NaOCH3, 38) and no parent peak was observed. HRMS (ESI) calcd. for C22H29O6
+: 389.1959 [M+H]+, found: 389.1963.
(R)-2-(1,4-dihydroxyl-4-methylpentyl)-1,4,5,8-tetramethoxynaphthalene (7)
Hydrolysis of 6 (1.5 g, 3.4 mmol) in 1 N sodium hydroxide (160 mL) and methanol (50 mL) was stirred at 0–5 °C for 12 h under a nitrogen atmosphere. Ethyl acetate was added to dilute the reactive mixture. Organic layer was washed with 4% HCl, water and saturated brine respectively, dried over anhydrous MgSO4 and evaporated to give the crude product, which was purified by column chromatography with ethyl acetate/petroleum ether (1/2, v/v) to produce 1.23 g of pale-yellow oil. Yield: 99%. [α]
25D
+ 143.7° (c 0.2, CHCl3). 1H NMR (300 MHz, CDCl3) δ: 7.02 (s, 1H, ArH), 6.81 (s, 2H, ArH), 5.24 (dd, 1H, J = 5.4, 5.1 Hz, CH), 3.92 (s, 9H, OCH
3), 3.72 (s, 3H, OCH
3), 1.95–1.54 (m, 4H, CH
2), 1.22 (s, 6H, CH
3). 13C NMR (75 MHz, CDCl3) δ: 152.4, 150.4, 149.2, 145.3, 133.4, 121.5, 119.2, 107.4, 106.7, 105.0, 69.5, 68.0, 61.7, 56.8, 56.1, 55.8, 39.1, 32.1, 28.7, 28.0. MS (ESI, %): 387 (M+Na+, 100), 419 (M++NaOCH3, 25), 751 (2M+Na+, 38) and no parent peak was observed. HRMS (ESI) calcd. for C20H29O6
+: 365.1959 [M+H]+, found: 365.1956.
(R)-2-(1-acetoxyl-4-hydroxyl-4-methylpentyl)-1,4,5,8-tetramethoxynaphthalene (8)
Acetic anhydride (10 mL) was added to a solution of 7 (1.20 g, 3.3 mmol) dissolved in pyridine (20 mL) at 0–5 °C, and the mixture was stirred for 2 h at the same temperature. Excess of the reagents were removed by HCl, NaHCO3, water and saturated brine in order, and then the crude product was purified by column chromatography with ethyl acetate/petroleum ether (1/1, v/v) to give 1.28 g of yellowish oil. Yield: 95%. [α]
25D
+ 145.7° (c 0.1, CHCl3). 1H NMR (300 MHz, CDCl3) δ: 6.86 (s, 1H, ArH), 6.83 (s, 2H, ArH), 6.36 (dd, 1H, J = 5.7, 6.0 Hz, CH), 3.93 (s, 6H, OCH
3), 3.88 (s, 3H, OCH
3), 3.83 (s, 3H, OCH
3), 2.11 (s, 3H, OCOCH
3), 2.04–1.25 (m, 4H, CH
2), 1.18 (s, 3H, CH
3), 1.17 (s, 3H, CH
3). 13C NMR (75 MHz, CDCl3) δ: 170.5, 153.7, 151.7, 150.4, 146.6, 131.1, 120.7, 120.6, 109.0, 107.9, 105.8, 71.3, 70.8, 62.7, 58.2, 57.6, 57.1, 39.6, 31.3, 29.9, 29.3, 21.6. MS (ESI, %): 429 (M+Na+, 100), 461 (M++NaOCH3, 15) and no parent peak was observed. HRMS (ESI) calcd. for C22H31O7
+: 407.2064 [M+H]+, found: 407.2067.
(R)-2-(1-acetoxyl-4-methyl-3-pentenyl)-1,4,5,8-tetramethoxynaphthalene (9)
Compound 8 (1.20 g, 2.96 mmol) in dry pyridine (50 mL) was cooled to −21 °C with ice-salted water, subsequently thionyl chloride was added. The reaction mixture was stirred at −21 °C for 15 min, and then poured into ice-water. The mixture was extracted with ethyl acetate twice, and organic layer combined was washed with water, saturated brine, and dried over anhydrous Na2SO4 and concentrated under reduced pressure. Column chromatography of the residue over alumina with ethyl acetate/petroleum ether (1/3, v/v) gave 945.8 mg of pale-yellow oil. Yield: 82.2%. [α]
25D
+124.5 (c 0.2, CHCl3). 1H NMR (300 MHz, CDCl3) δ: 6.87 (s, 1H, ArH), 6.82 (s, 2H, ArH), 6.34 (dd, 1H, J = 4.5, 6.0 Hz, CH), 6.15 (t, 1H, J = 4.5 Hz, CH), 3.93 (s, 6H, OCH
3), 3.86 (s, 3H, OCH
3), 3.83 (s, 3H, OCH
3), 2.59–2.54 (m, 2H, CH
2), 2.10 (s, 3H, OCOCH
3), 1.65 (s, 3H, CH
3), 1.55 (s, 3H, CH
3). 13C NMR (75 MHz, CDCl3) δ: 170.4, 153.5, 151.6, 150.8, 147.1, 134.8, 130.9, 122.9, 120.9, 119.4, 109.0, 108.2, 105.6, 71.1, 62.7, 58.1, 57.7, 57.3, 34.8, 25.9, 21.5, 18.1. MS (ESI, %): 411 (M+Na+, 100), 443 (M++NaOCH3, 18) and no parent peak was observed. HRMS (ESI) calcd. for C22H28O6Na+: 411.1778 [M+Na]+, found: 411.1776.
(R)-2-(1-hydroxyl-4-methyl-3-pentenyl)-1,4,5,8-tetramethoxynaphthalene (10)
Hydrolysis of 9 (1 g, 2.6 mmol) in 1 N sodium hydroxide (100 mL) and methanol (50 mL) was stirred at 0–5 °C for 12 h under a nitrogen atmosphere. Ethyl acetate was added to dilute the reactive mixture. Organic layer was washed with water and saturated brine, and dried over anhydrous MgSO4, and then evaporated under reduced pressure. The crude product was purified by column chromatography over silica gel with ethyl acetate/petroleum ether (1/4, v/v) to obtain 839.2 mg of desirable compound. Yield: 94%. [α]
25D
+149.2° (c 0.24, CHCl3). 1H NMR (300 MHz, CDCl3) δ: 7.02 (s, 1H, ArH), 6.82 (s, 2H, ArH), 5.33 (m, 2H, CH, CH), 3.95 (s, 6H, OCH
3), 3.93 (s, 3H, OCH
3), 3.76 (s, 3H, OCH
3), 2.55–2.51 (m, 2H, CH
2), 1.72 (s, 3H, CH
3), 1.65 (s, 3H, CH
3). 13C NMR (75 MHz, CDCl3) δ: 153.6, 151.7, 150.5, 146.8, 135.4, 134.2, 122.9, 120.5, 108.6, 108.1, 106.4, 68.8, 63.0, 58.6, 58.1, 57.4, 57.2, 37.4, 25.1, 18.2. MS (ESI, %): 369 (M+Na+, 100), 401 (M++NaOCH3, 38) and no parent peak was observed. HRMS (ESI) calcd. for C20H27O5
+: 347.1853 [M+H]+, found: 347.1856.
(R)-4-methyl-1-(1,4,5,8-tetramethoxynaphthalen-2-yl)pent-3-en-1-yl-4-((tertbutoxycarbonyl)amino) benzoate (11)
To a stirred solution of 10 (2.0 g, 5.8 mmol) and 4-((tertbutoxycarbonyl)amino)benzoic acid (1.66 g, 7.0 mmol) in anhydrous DCM were added DCC (1.4 g, 7.0 mmol) and DMAP (350 mg, 2.9 mmol). After stirring overnight at room temperature, petroleum ether was added into the reaction mixture to facilitate precipitates at 4 °C, and then the solution was filtered, and concentrated in vacuo. The residue was purified by flash chromatography to afford 2.99 g of 11 as colorless oil. Yield: 91%. [α] D25 +139.7° (c 0.25, CHCl3). 1H NMR (400 MHz, CDCl3) δ: 7.93 (d, 2H, J = 0.8 Hz, ArH), 7.37 (d, 2H, J = 0.8 Hz, ArH), 6.86 (s, 1H, ArH), 6.73 (s, 2H, ArH), 6.42–6.47 (m, 1H, CH), 5.14 (t, J = 7.2 Hz, 1H, CH), 3.85 (s, 3H, OCH
3), 3.82 (s, 3H, OCH
3), 3.78 (s, 6H, OCH
3), 2.55–2.67 (m, 2H, CH
2), 1.56 (s, 3H, CH
3), 1.49 (s, 3H, CH
3), 1.42 (s, 9H, CH
3). 13C NMR (100 MHz, CDCl3) δ: 164.6, 152.1, 151.0, 150.4, 149.4, 145.5, 141.6, 133.5, 130.6, 129.7, 123.6, 121.5, 119.3, 118.1, 116.3, 107.4, 106.4, 104.8, 80.0, 70.3, 61.4, 56.7, 56.1, 55.9, 33.6, 27.1, 24.7, 17.0. HRMS (ESI), calcd. for C32H40NO8
+: 566.2748 [M+H]+, found: 566.2744.
(R)-6-(1-(4-(N-(tertbutoxycarbonyl)amino)benzoyloxy)-4-methylpent-3-en-1-yl)-5,8-dimethoxy-1,4-naphthoquinone (12)
A solution of CAN (3.69 g, 6.8 mmol) in water (20 mL) was added dropwise to a stirred solution of 11 (3.28 g, 5.8 mmol) in the ice bath. The mixture was risen up to room temperature, and stirred for additional 10 min, and then diluted with water and ethyl acetate. Organic layer was separated and aqueous layer was extracted with ethyl acetate (2 × 100 mL). The combined organic extracts were washed with saturated brine (150 mL), and dried over anhydrous Na2SO4, and then concentrated under reduced pressure. The residue was purified by column chromatography with ethyl acetate/petroleum ether (1/1, v/v) to give 3.1 g of compound 12 as yellow oil. Yield: 91%, 1H NMR (400 MHz, CDCl3) δ: 7.94 (d, J = 0.8 Hz, 2H, ArH), 7.42 (d, J = 0.8 Hz, 2H, ArH), 7.23 (s, 1H, ArH), 6.70 (s, 2H, QuinoneH), 6.22 (t, J = 4.0 Hz, 1H, CH), 5.14 (t, J = 6.8 Hz, 1H, CH), 3.91 (s, 3H, OCH
3), 3.80 (s, 3H, OCH
3), 2.59–2.64 (m, 1H, CH
2
), 2.49–2.56 (m, 1H, CH
2
), 1.61 (s, 3H, CH
3), 1.50 (s, 3H, CH
3), 1.44 (s, 9H, CH
3). 13C NMR (100 MHz, CDCl3) δ: 184.8, 184.3, 165.3, 156.1, 152.2, 150.6, 144.9, 143.2, 138.9, 137.8, 135.8, 130.8, 125.2, 123.9, 120.1, 118.2, 117.5, 116.6, 81.3, 71.2, 62.0, 56.6, 34.1, 28.2, 25.8, 17.9. HRMS (ESI) calcd. for C30H34NO8
+: 536.2279 [M+H]+, found: 536.2284.
(R)-5,8-dimethoxyl-6-(1-hydroxyl-4-methylpentyl)-1,4-naphthaquinones (13)
A solution of K2CO3 (6.6 g, 48.0 mmol) was added dropwise to a stirred solution of 12 (12.9 g, 24.0 mmol) dissolved in THF (250 mL) at ice-bath. The reaction mixture was stirred for 2 h at the same temperature. The progression was monitored by TLC. After completion, the mixture was neutralized with statured NH4Cl solution, and then diluted with water and ethyl acetate. Organic layer was separated and aqueous layer was extracted with ethyl acetate (2 × 150 mL). The combined organic extracts were washed with saturated brine (200 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography with ethyl acetate/petroleum ether (1/1, v/v) as an eluent to give 6.98 g of yellowish oil 13. Yield: 92%. [α]
25D
+48.5° (c 0.5, CHCl3). 1H NMR (300 MHz, CDCl3) δ: 7.55 (s, 1H, ArH), 6.79 (d, 2H, J = 3.0 Hz, QuinoneH), 5.24 (t, 1H, J = 6.0 Hz, CH), 5.10 (t, 1H, J = 3.0 Hz, CH), 3.97 (s, 3H, OCH
3), 3.89 (s, 3H, OCH
3), 2.35–2.19 (m, 2H, CH
2), 1.76 (s, 3H, CH
3), 1.65 (s, 3H, CH
3). 13C NMR (75 MHz, CDCl3) δ: 185.1, 184.5, 156.5, 150.9, 147.9, 139.2, 137.9, 136.9, 125.1, 68.8, 62.4, 56.9, 37.2, 26.1, 18.2. MS (ESI, %): 317 (M+H+, 12.5), 339 (M+Na+, 30), 371 (M++NaOCH3, 100). HRMS (ESI) calcd. for C18H20O5Na+: 339.1203 [M+Na]+, found: 339.1207.
(R)-1-(1,4-dimethoxy-5,8-dioxo-5,8-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl 3-hydroxy-3-methylbutanoate (14a)
To a stirred solution of 13 (3.16 g, 10.0 mmol) and 3-hydroxy-3-methylbutanoic acid (1.30 g, 11.0 mmol) in anhydrous DCM were added DCC (2.27 g, 11.0 mmol) and DMAP (350 mg, 2.9 mmol). TLC was applied to monitor the progression. After completion, petroleum ether was added into the reaction mixture to facilitate precipitates at 4 °C, and filtered to remove the insoluble substance, and concentrated in vacuo. The residue was purified by flash chromatography to afford 2.54 g of 14a as yellow oil. Yield: 61%. [α]
25D
+59.3° (c 0.4, CHCl3). 1H NMR (300 MHz, CDCl3) δ: 7.27 (s, 1H, ArH), 6.67 (d, 2H, J = 3.0 Hz, QuinoneH), 6.18 (m, H, CH), 5.04 (t, 1H, J = 8.1 Hz, CH), 3.95 (s, 3H, OCH
3), 3.94 (s, 3H, OCH
3), 2.58–2.38 (m, 4H, 2 × CH
2), 1.68 (s, 3H, CH
3), 1.55 (s, 3H, CH
3), 1.29 (s, 3H, CH
3), 1.26 (s, 3H, CH
3). 13C NMR (75 MHz, CDCl3) δ: 187.6, 186.5, 173.2, 152.1, 138.7, 134.2, 132.0, 124.1, 119.7, 115.2, 114.3, 70.9, 70.0, 62.3, 55.4, 42.1, 32.4, 29.2, 24.4, 18.1. HRMS (ESI): calcd for C23H29O7
+: 417.1908 [M+H]+, found: 417.1902. These data were in accordance with the literature [8].
(R)-1-(1,4-dimethoxy-5,8-dioxo-5,8-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl tetrahydrofuran-3-carboxylate (14b)
The preparation procedure for compound 14b was similar to that of compound 14a, and tetrahydrofuran-3-carboxylic acid was substituted for 3-hydroxy-3-methylbutanoic acid. Yield: 71%. [α]
25D
+56.3° (c 0.5, CHCl3). 1H NMR (300 MHz, CDCl3) δ: 7.24 (d 1H, J = 3.0 Hz, ArH), 6.78 (d, 2H, J = 3.3 Hz, QuinoneH), 6.16 (m, 1H, CH), 5.11 (t, 1H, J = 6.3 Hz, CH), 4.02–3.79 (m, 10H, 2 × OCH
3, 2 × OCH
2), 3.19 (m, 1H, CH), 2.53–2.44 (m, 2H, CH
2), 1.68 (s, 3H, CH
3), 1.54 (s, 3H, CH
3). 13C NMR (75 MHz, CDCl3) δ: 186.3, 186.2, 173.2, 152.3, 152.2, 138.7, 134.1, 132.2, 120.1, 119.8, 114.3, 114.2, 75.6, 70.9, 70.5, 61.8, 55.4, 42.5, 32.3, 31.6, 24.5, 18.3. HRMS (ESI) calcd for C23H27O7
+: 415.1751 [M+H]+; found: 415.1756. These data were in accordance with the literature [8].
(R)-1-(1,4-dimethoxy-5,8-dioxo-5,8-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl furan-3-carboxylate (14c)
The preparation procedure of compound 14c was similar to that of compound 14a, 3-hydroxy-3-methylbutanoic acid was replaced with furan-3-carboxylic acid. Yield: 55%. [α]
25D
+48.3° (c 0.3, CHCl3). 1H NMR (300 MHz, CDCl3) δ: 8.10 (d, 1H, J = 1.2 Hz, FuranylH), 7.49 (d, 1H, J = 1.2 Hz, FuranylH), 7.29 (s, 1H, ArH), 6.82 (d, 2H, J = 3.0 Hz, QuinoneH), 6.80 (s, 1H, FuranylH), 6.52 (dd, 1H, J = 4.8, 4.8 Hz, CH), 5.19 (t, 1H, J = 7.5 Hz, CH), 3.97 (s, 3H, OCH
3), 3.94 (s, 3H, OCH
3), 2.63–2.57 (m, 2H, CH
2), 1.69 (s, 3H, CH
3), 1.58 (s, 3H, CH
3). 13C NMR (75 MHz, CDCl3) δ: 187.1, 186.9, 159.2, 152.1, 148.6, 143.9, 137.6, 137.5, 134.2, 132.1, 119.7, 119.6, 118.3, 114.9, 114.4, 110.6, 70.1, 62.4, 55.8, 32.2, 24.4, 18.3. HRMS (ESI): calcd. for C23H23O7
+: 411.1438 [M+H]+, found: 411.1442. These data were in accordance with the literature [8].
Chiral HPLC analysis conditions for shikonin and its derivatives
The chiral HPLC column applied (150 × 4.6 mm) was Sino-Chiral OD [No. 0A02014-C (Packing cellulose-tris (3,5-dimethylphenyl carbamate)], which was purchased from FunSea Beijing Technology Co. Ltd (Beijing). All the separations were performed at ambient temperature. The mobile phase, hexane–isopropanol (80:20, v/v) was degassed before application. To obtain sufficient resolution of shikonin, alkannin and their derivatives, the flow rate of mobile phase was adjusted to 0.65 mL/min and injection volume was set at 5 μL.