Anti-Inflammatory Isoquinoline with Bis-seco-aporphine Skeleton from Dactylicapnos scandens

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1 Anti-Inflammatory Isoquinoline with Bis-seco-aporphine Skeleton from Dactylicapnos scandens Bei Wang,,, Zi-Feng Yang,, Yun-Li Zhao, Ya-Ping Liu, Jun Deng, Wan-Yi Huang, Xiao-Nian Li, Xin-Hua Wang *, and Xiao-Dong Luo *,, State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming , People s Republic of China Guangzhou Medical University, Guangzhou , People s Republic of China University of Chinese Academy of Sciences, Beijing , People s Republic of China Supporting Information Contents of Supporting Information 1. Supplementary Figures Figure S01. 1 H NMR spectrum of 1 in CDCl Figure S C NMR and DEPT spectra of 1 in CDCl Figure S03. HSQC spectrum of 1 in CDCl Figure S04. HMBC spectrum of 1 in CDCl Figure S05. HRESIMS spectrum of Figure S06. ESIMS spectrum of Figure S07. UV spectrum of Figure S08. Effect of 1 on the cell viability in RAW cells after 24 h treatment Crystal data and structure refinement for dactyllactone A (1) Experimental procedures General experimental procedures Plant material Cytotoxic Assay

2 1. Supplementary Figures Figure S01. 1 H NMR spectrum of 1 in CDCl 3 2

3 Figure S C NMR and DEPT spectra of 1 in CDCl 3 3

4 Figure S03. HSQC spectrum of 1 in CDCl 3 4

5 Figure S04. HMBC spectrum of 1 in CDCl 3 5

6 Figure S05. HRESIMS spectrum of 1 6

7 Figure S06. ESIMS spectrum of 1 7

8 Figure S07. UV spectrum of 1 8

9 2. Crystal data and structure refinement for dactyllactone A (1). project data Identification code cu_wd5a_0m Empirical formula C21 H21 N O8 Formula weight Temperature 100(2) K Wavelength Å Crystal system Monoclinic Space group P21/c Unit cell dimensions a = (5) Å = 90. b = (7) Å = (2). c = (5) Å = 90. Volume (2) Å3 Z 8 Density (calculated) Mg/m3 Absorption coefficient mm-1 F(000) 1744 Crystal size x x mm3 Theta range for data collection to Index ranges -13<=h<=16, -22<=k<=23, -16<=l<=17 Reflections collected Independent reflections 6874 [R(int) = ] Completeness to theta = % Absorption correction Semi-empirical from equivalents Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 6874 / 0 / 551 Goodness-of-fit on F Final R indices [I>2sigma(I)] R1 = , wr2 = R indices (all data) R1 = , wr2 = Extinction coefficient n/a Largest diff. peak and hole and e.å-3 X-ray crystallographic data for compound 1 is available from the Cambridge Crystallographic Data Centre (CCDC ). These data can be obtained free of charge ( ). 9

10 3. Experimental procedures. 3.1 General experimental procedures X-ray crystallography was performed on a Bruker APEX DUO diffractometer (Bruker Ltd, Karlsruhe, Germany) using graphic-monochromated Cu Kα radiation. Melting point was obtained with a WRX-4 micromelting point instrument (Shanghai instrument equipment Co. Ltd., P.R. China). UV spectrum was measured with a Shimadzu UV2401PC spectrometer. 1 H, 13 C, and 2D NMR spectra were recorded on a Bruker DRX-600 spectrometer with TMS as an internal standard. Unless otherwise specified, chemical shifts (δ) were expressed in ppm with reference to solvent signals. ESIMS analyses was carried out on Agilent 1290 UPLC/6540 Q-TOF mass spectrometers. HREIMS analyses was carried out on Waters AutoSpec Premier P776 mass spectrometers. Column chromatography (CC) was performed on silica gel ( and mesh, Qingdao Marine Chemical Co. Ltd., P.R. China), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB). Fractions were monitored by TLC (GF 254, Qingdao Marine Chemical Co., Ltd., P. R. China) and spots were visualized by Dragendorff s reagent. 3.2 Plant material Air-dried roots of D. scandens were purchased from a market of Chinese medical materials located at Zhonghao-Luoshi-Wan of Kunming, Yunnan province, P.R. China, in February 2015.The material was identified by Dr. Ya-Ping Liu. A voucher specimen (No ) has been deposited at Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China. 3.3 Cytotoxic Assay The RAW264.7 cells used in the cytotoxic activity assay were purchased from (ATCC, Manassas, VA, USA). All cells were cultured in Dulbecco s modified Eagle s 10

11 medium (DMEM) (Hyclone, USA) supplemented with 10% fetal bovine serum (Hyclone, USA) in a humidified environment with 5% CO2 at 37. Briefly, cells were harvested and diluted with complete medium into density of cells/ml. 200µL suspended cells were seeded into 96-well plates. 24 h later, medium was gently removed, and 200 µl new medium with dactyllactone A (i.e. 12, 2.4, and 0.48 µm) were added into each well. At the end of the treatment with dactyllactone A for 24 h, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to plates and they were incubated for another 4 h. The medium was then carefully removed; 200 μl DMSO was added into each well and the plates were shook in the shadow for 15 minutes. OD values were determined by a Flex Station 3 Multi-Mode Microplate Reader (Molecular Devices) at 492 nm. Inhibitory ratios were calculated using the following formula: Corrected OD value = OD value of sample group OD value of the blank control group. The inhibitory ratio (%) = [(corrected OD value of DMSO control group- corrected OD value of sample group)/corrected OD value of DMSO control group] 100%. Figure S08. Effect of dactyllactone A on the cell viability in RAW cells after 24 h treatment. These data are expressed as the mean ± SEM. 11