Taxodikaloids A and B, Two Dimeric Abietane-Type Diterpenoids from Taxodium ascendens Possessing an Oxazoline Ring Linkage

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1 Supporting Information for Taxodikaloids A and B, Two Dimeric Abietane-Type Diterpenoids from Taxodium ascendens Possessing an Oxazoline Ring Linkage Xing-Hao Huang,, Ling-Xue Tao, Chang-Qiang Ke, Chunping Tang, Hai-Yan Zhang, Yang Ye,, Li-Gen Lin, *, and Sheng Yao *, State Key Laboratory of Drug Research & Natural Products Chemistry Department, Shanghai Institute of Materia Medica, and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu-Chong-Zhi Road, Zhangjiang Hi-Tech Park, Shanghai , China University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing , China School of Life Science and Technology, ShanghaiTech University, Shanghai , China State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China 1

2 Table of Contents EXPERIMENTAL SECTION... 3 Table S1. X-ray crystallographic data for Table S2. Neuroprotective effect of compounds 1 and 2 against Aβ induced damage in SH-SY5Y cells Figure S1. 1 H NMR (600 MHz) spectrum of taxodikaloid A (1) in CDCl Figure S2. 13 C and DEPT NMR (150 MHz) spectra of taxodikaloid A (1) in CDCl Figure S3. 1 H- 1 H COSY (600 MHz) spectrum of taxodikaloid A (1) in CDCl Figure S4. HSQC (600 MHz) spectrum of taxodikaloid A (1) in CDCl Figure S5. HMBC (600 MHz) spectrum of taxodikaloid A (1) in CDCl Figure S6. NOESY (600 MHz) spectrum of taxodikaloid A (1) in CDCl Figure S7. IR spectrum of taxodikaloid A (1) Figure S8. EI-MS data of taxodikaloid A (1) Figure S9. HR EI-MS data of taxodikaloid A (1) Figure S10. 1 H NMR (500 MHz) spectrum of taxodikaloid B (2) in CDCl Figure S C and DEPT NMR (125 MHz) spectra of taxodikaloid B (2) in CDCl Figure S12. 1 H- 1 H (500 MHz) COSY spectrum of taxodikaloid B (2) in CDCl Figure S13. HSQC (500 MHz) spectrum of taxodikaloid B (2) in CDCl Figure S14. HMBC (500 MHz) spectrum of taxodikaloid B (2) in CDCl Figure S15. ROESY (400 MHz) spectrum of taxodikaloid B (2) in CDCl Figure S16. IR spectrum of taxodikaloid B (2) Figure S17. ESI-MS data of taxodikaloid B (2) Figure S18. HR ESI-MS data of taxodikaloid B (2)

3 EXPERIMENTAL SECTION General Experimental Procedures Optical rotations were obtained with a Rudulph Autopol VI Automatic polarimeter. UV spectra were messured on a Shimadzu UV-2550 UV/Visible spectrophotometer. ECD spectra were recorded on a JASCO J-810 spectrometer. IR spectra were detected on a Nicolet Magna FT-IR 750 spectrophotometer with KBr pellets. Melting point was messured on a XT-4 melting point apparatus. 1D and 2D NMR spectra were record on a Bruker Avance III 400 (Bruker, Ettlingen, Germany), a Bruker Avance III 500 (Bruker, Ettlingen, Germany), a Bruker Avance III 600 (Bruker, Ettlingen, Germany) and a Varian MR-400 NMR spectrometers with TMS as internal standard. The chemical shift (δ) values are given in ppm with reference to the solvent signals, and coupling constants (J) are in Hz. HR EI-MS and HR ESI-MS data were recorded on a Finnigan MAT95 and a Thermo Fisher Scientific ORBITRAP-ELITE mass spectrometers. Silica gel (Qingdao Marine Chemical Industrials, Qingdao, People s Republic of China), MCI gel CHP20/P120 (Mitsubishi Chemical Corporation, Tokyo, Japan) and Sephadex LH-20 (GE healthcare, Uppsala, Sweden) were used for column chromatography (CC). TLC was carried out on Silica gel 60 F 254 (Merck KGaA, Darmstadt, Germany) and the TLC spots were viewed at 254 nm and visualized by heating silica gel plates sprayed with 5 % H 2 SO 4 in EtOH containing 10 mg/ml vanillin. Analytical HPLC and ESI-MS spectra were performed on a Waters e2695 Separations Module with a 2998 PDA coupled with a Waters 2424 ELSD detector. Preparative HPLC was performed on a Varian PrepStar SD-1 system with an Alltech 3300 ELSD detector. Semi-preparative HPLC was performed on a Waters 2690 Separations Module with a Waters 996 Photodiode Array Detector. Chromatographic separations were carried out on a Waters Sunfire RP C18, 5 μm, mm column and a YMC-Pack ODS-A, S-5 μm, 12 nm, mm column, using a gradient solvent system composed of H 2 O and CH 3 CN, with a flow rate of 10.0 and 3.0 ml/ min, respectively. All solvents used for CC and HPLC were of analytical grade (Shanghai Chemical Reagents Co., Ltd.) and gradient grade (Merck KGaA, Germany), respectively. 3

4 Plant Material The seeds of Taxodium ascendens were collected locally in SheXian, Anhui province, People s Republic of China, in November 2012, and identified by Professor Jin-Gui Shen of Shanghai Institution of Materia Medica. A voucher specimen (No ) has been deposited at the Herbarium of Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Isolation process The air-dried seeds of T. ascendens (11 kg) were ground and extracted with 95% ethanol thrice (7 days each). The pooled extracts were concentrated under reduced pressure to yield a residue (1.8 kg), which was then suspended in hot water (5 L) and extracted with petroleum ether (PE) and EtOAc, successively, yielding a PE (380 g) and an EtOAc (150 g) fraction. The EtOAc fraction was separated by column chromatography (CC) over MCI gel, eluted with EtOH in H 2 O (40%, 60%, 80%, and 95%, v/v), to yield four fractions (Fr.1 Fr.4). Fr.3 was subjected to CC over silica gel ( mesh), eluted with PE EtOAc (20:1, 15:1, 10:1, 8:1, 5:1, 2:1, and 1:2, v/v), giving 12 fractions (3A 3L). Fraction 3I was subjected to CC over a Sephadex LH-20 gel column, eluted with CHCl 3 MeOH (1:1, v/v), to obtain subfractions 3I1 3I4. Subfraction 3I2 was then applied to a silica gel column ( mesh), eluted with PE acetone (10:1 to 1:1, v/v), to yield eight subfractions 3I2A 3I2H. Subfraction 3I2D was further purified by preparative HPLC (CH 3 CN H 2 O, 70 90%, 0 90 min) and then semi-preparative HPLC (CH 3 CN H 2 O, 70 95%, 0 60 min) to give compounds 1 (5.3 mg) and 2 (8.6 mg) (Figure 1). Compound Characterization Taxodikaloid A (1): green needle crystals; [α] 20 D (c 0.267, MeOH); UV (MeOH) λ max (log ε) 220 (4.15), 228 (3.99), 250 (3.73) nm; ECD (MeOH) 202 nm (Δε +1.86), 224 nm (Δε -0.50), 248 nm (Δε +2.52), 285 nm (Δε +1.08), 340 nm (Δε -0.97); IR (KBr) ν max : 3424, 2965, 2920, 2853, 1739, 1265, 1102, 1030, 800 cm -1 ; 1 H and 13 C NMR (CDCl 3 ) data, see Table 1; HREIMS m/z [M] + (calcd for C 40 H 49 NO 6, ); melting point C. Taxodikaloid B (2): dark green amorphous powder; [α] 20 D (c 0.087, MeOH); UV (MeOH) λ max (log ε) 237 (4.16), 291 (3.99), 339 (3.97) nm; ECD (MeOH) 208 nm (Δε +6.62), 231 nm (Δε -0.94), 246 nm (Δε +2.81), 262 nm (Δε -0.23), 292 nm (Δε +2.26), 330 nm (Δε

5 -1.56); IR (KBr) ν max : 3429, 2962, 2928, 1736, 1663, 1606, 1465, 1292 cm -1 ; 1 H and 13 C NMR (CDCl 3 ) data, see Table 1; (+)-HRESIMS m/z [M + H] + (calcd for C 40 H 54 NO 6, ). X-ray Crystallographic Analyses Green needle crystals of 1 were obtained by recrystallization from MeOH. The crystal data was collected on a Bruker D8 Venture diffractometer using graphite-monochromated Cu Kα radiation. The structure of 1 was solved by direct methods using SHELXS-97. Refinements were performed with SHELXL-2013 using fullmatrix least-squares calculations on F 2, with anisotropic displacement parameters for all the nonhydrogen atoms. The hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms. The Crystallographic data have been deposited at the Cambridge Crystallographic Data Center (deposition no. CCDC ), which can be obtained free of charge from the CCDC via Neuroprotective Assay Human neuroblastoma SH-SY5Y cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained at 37 C in a humidified atmosphere containing 5% CO 2. Cells were seeded into 96-well plates and 6-well plates (Corning, Corning, NY, USA) at a density of cells/ml in MEM/F12 medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY), 100 U/mL penicillin, 100 μg/ml streptomycin, and 2 mm L -glutamine. Cells were used at 24 hours after seeded. Drug treatment and A exposure: the culture medium of the cells was replaced with serum-free MEM/F12, and the cells were pretreated with each compound (1 or 10 μm) or epigallocatechin gallate (EGCG, 10 μm) for 2 hours, followed by exposure to 10 μm of Aβ (Sigma) in the presence of compounds for another 24 hours. Cell viability test: Cell viability was evaluated by morphological observation under microscope (Nikon TE2000, Melville, NY, USA) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, St Louis, MO, USA) assay. Twenty-four hours after cells were treated with A, MTT was added to culture medium at a final concentration of 0.5 mg/ml and incubated at 37 C for 4 hours. When 5

6 obtained by the live cell, MTT is transformed to formazan. The formazan product was dissolved by adding 100 μl DMSO each well. Cell plates were shaken for 5 minutes and the absorbance of each well was recorded on a DTX 800 Multimode Detector (Beckman Coulter, Fullerton, CA) at 490 nm. 6

7 Table S1. X-ray crystallographic data for 1 Identification code Empirical formula Formula weight Temperature Wavelength Crystal system yws_0m_pl C40 H49 N O6 296 K Å Orthorhombic Space group P Unit cell dimensions a = (3) Å a= 90. b = (7) Å b= 90. c = (2) Å g = 90. Volume (9) Å 3 Z 2 Density (calculated) Mg/m 3 Absorption coefficient mm -1 F(000) 688 Crystal size 0.39 x 0.32 x 0.3 mm 3 Theta range for data collection to Index ranges Reflections collected <=h<=14, -28<=k<=28, -7<=l<=6 Independent reflections 3093 [R(int) = ] Completeness to theta = % Absorption correction Semi-empirical from equivalents Max. and min. transmission and Refinement method Full-matrix least-squares on F 2 Data / restraints / parameters 3093 / 16 / 252 Goodness-of-fit on F Final R indices [I>2sigma(I)] R1 = , wr2 = R indices (all data) R1 = , wr2 = Absolute structure parameter 0.21(8) Extinction coefficient Largest diff. peak and hole and e.å -3 n/a 7

8 Table S2. Neuroprotective effect of compounds 1 and 2 against Aβ induced damage in SH-SY5Y cells. Compounds Cell viability a (%) (n = 4) 10 μm 1 μm % ± 3.35% ** 67.20% ± 3.83% % ± 3.31%** 74.42% ± 5.70% EGCG % ± 5.82%** N.T. b a The neuroprotective effect of the isolated was evaluated on Aβ induced neurotoxicity in SH-SY5Y cells. The cell viability in control was considered as 100%, and the cell viability under Aβ exposure was 63.15% ± 0.55%. The positive control is epigallocatechin gallate (EGCG). b N.T. means not tested. ** p<0.01 8

9 Figure S1. 1 H NMR (600 MHz) spectrum of taxodikaloid A (1) in CDCl3. 9

10 Figure S2. 13 C and DEPT NMR (150 MHz) spectra of taxodikaloid A (1) in CDCl 3. 10

11 Figure S3. 1 H- 1 H COSY (600 MHz) spectrum of taxodikaloid A (1) in CDCl 3. 11

12 Figure S4. HSQC (600 MHz) spectrum of taxodikaloid A (1) in CDCl 3. 12

13 Figure S5. HMBC (600 MHz) spectrum of taxodikaloid A (1) in CDCl 3. 13

14 Figure S6. NOESY (600 MHz) spectrum of taxodikaloid A (1) in CDCl 3. 14

15 Figure S7. IR spectrum of taxodikaloid A (1). 15

16 Figure S8. EI-MS data of taxodikaloid A (1) 16

17 Figure S9. HR EI-MS data of taxodikaloid A (1). 17

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19 Figure S10. 1 H NMR (500 MHz) spectrum of taxodikaloid B (2) in CDCl 3. 19

20 Figure S C and DEPT NMR (125 MHz) spectra of taxodikaloid B (2) in CDCl 3. 20

21 Figure S12. 1 H- 1 H (500 MHz) COSY spectrum of taxodikaloid B (2) in CDCl 3. 21

22 Figure S13. HSQC (500 MHz) spectrum of taxodikaloid B (2) in CDCl 3. 22

23 Figure S14. HMBC (500 MHz) spectrum of taxodikaloid B (2) in CDCl 3. 23

24 Figure S15. ROESY (400 MHz) spectrum of taxodikaloid B (2) in CDCl 3. 24

25 Figure S16. IR spectrum of taxodikaloid B (2). 25

26 Figure S17. ESI-MS data of taxodikaloid B (2). 26

27 Figure S18. HR ESI-MS data of taxodikaloid B (2). 27

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