Supporting Information

Supporting Information Pepluanols C-D, Two Diterpenoids with Two Skeletons from Euphorbia peplus Luo-Sheng Wan,,ǁ, Yin Nian,, Xing-Rong Peng, Li-Dong Shao, Xiao-Nian Li, Jian Yang, Ming Zhou,,,* and Ming-Hua Qiu.*, State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, P. R. China Key Laboratory of Animal Models and Human Diease Mechanisms, and Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, P. R. China Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, United States ǁ Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Pharmacy Department of Tongji Medical School, Huazhong University of Science and Technology, Wuhan 430030, P. R. China 1

Table of Contents Table S1. 1 H, 1 H- 1 H COSY, and HMBC NMR data of pepluanol C (1)... 3 Table S2. 1 H, 1 H- 1 H COSY, and HMBC NMR data of pepluanol D (2)... 3 Figure S1. Effect of compounds 1 and 2 on N-type (Ca v 2.2), T-type (Ca v 3.1), and L-type (Ca v 1.2) calcium channels.... 4 Figure S2. Effect of compounds 1 and 2 on herg..... 4 Experimental section General experimental procedures... 5 Plant material... 5 Extraction and isolation... 5 Physical constants and spectral data of 1 2... 6 Bioassay methods... 6 X-ray crystallographic data and deposition information... 7 Figure S3: 1 H NMR spectrum of Pepluanol C (1) in CDCl 3... 9 Figure S4: 13 C NMR and DEPT spectra of Pepluanol C (1) in CDCl 3... 10 Figure S5: HSQC spectrum of Pepluanol C (1) in CDCl 3... 11 Figure S6: HMBC spectrum of Pepluanol C (1) in CDCl 3... 12 Figure S7: 1 H- 1 H COSY spectrum of Pepluanol C (1) in CDCl 3... 13 Figure S8: ROESY spectrum of Pepluanol C (1) in CDCl 3... 14 Figure S9: Specific ROESY correlations from OH-4 to H-13 of Pepluanol C (1) in CDCl 3... 15 Figure S10: HR-ESI (+) MS spectrum of Pepluanol C (1)... 16 Figure S11: CD spectrum of Pepluanol C (1) in MeOH... 17 Figure S12: 1 H NMR spectrum of Pepluanol D (2) in CDCl 3... 18 Figure S13: 13 C NMR and DEPT spectra of Pepluanol D (2) in CDCl 3... 19 Figure S14: HSQC spectrum of Pepluanol D (2) in CDCl 3... 20 Figure S15: HMBC spectrum of Pepluanol D (2) in CDCl 3... 21 Figure S16: 1 H- 1 H spectrum of Pepluanol D (2) in CDCl 3... 22 Figure S17: ROESY spectrum of Pepluanol D (2) in CDCl 3... 23 Figure S18: HR-ESI (+) MS spectrum of Pepluanol D (2)... 24 Figure S19: CD spectrum of Pepluanol D (2) in MeOH... 25 2

Table S1. 1 H, 1 H- 1 H COSY, and HMBC NMR data of pepluanol C (1) (recorded at 600 MHz in CDCl 3, J Hz) H 1 H NMR 1 H- 1 H COSY HMBC 1 5.65 brs C-2, C-3, C-4, C-14, C-15, C-16 3 5.26 s C-1, C-2, C-4, C-5, C-15, C-16, C-1 5 3.52 s C-3, C-4, C-6, C-7, C-17 7 3.35 brt; 1.74 m H-8 C-5, C-6, C-8, C-9, C-14, C-17 8 5.21 m H 2-7, H-9 C-7, C-10 9 5.56 d (10.9) H-8 C-7, C-10, C-11, C-18 11 5.34 d (16.6) H-12 C-13, C-18, C-19 12 6.19 m H-11, H-13 C-10, C-20 13 2.53 m H-12, H 3-20 C-11, C-12, C-14, C-15, C-20 16 1.74 brs C-1, C-2, C-3 17 1.07 s C-5, C-6, C-7, C-14 18 1.07 s C-9, C-10, C-11, C-19 19 1.19 s C-9, C-10, C-11, C-18 20 1.35 d (4.3) H-13 C-12, C-13, C-15 OH-4 3.26 s C-3, C-4, C-5, C-15 Table S2. 1 H, 1 H- 1 H COSY, and HMBC NMR data of pepluanol D (2) (recorded at 600 MHz in CDCl 3, J Hz) H 1 H NMR 1 H- 1 H COSY HMBC 1 6.42 brs H-15 C-2, C-3, C-13, C-14, C-15, C-16 5 4.70 s C-3, C-6, C-7 7 5.17 brs H-8 C-5, C-6, C-8, C-9, C-14, C-17 8 2.31 m H-7, H-9 C-6, C-7, C-9, C-10, C-14, C-15 9 0.43 dd (11.5, 8.7) H-8, H-11 C-7, C-8, C-10, C-11, C-12, C-14, C-18, C-19 11 0.83 ddd (11.5, 8.7, 6.6) H-9, H-12 C-8, C-9, C-10, C-12, C-13, C-18, C-19 12 2.00 m; 1.47 m H-11, H-13 C-9, C-11, C-13, C-15 13 2.28 m H-12, H-15, H 3-20 C-14, C-15 15 2.92 m H-1, H-13 C-1, C-2, C-8, C-13, C-14, C-20 16 1.86 d (1.0) C-1, C-2, C-3 17 1.80 brs C-5, C-6, C-7 18 1.14 s C-9, C-10, C-11, C-19 19 1.14 s C-9, C-10, C-11, C-18 20 1.20 d (7.6) H-13 C-12, C-13, C-15 OH-4 3.66 s C-4, C-14, C-5 OH-5 3.65 s C-4, C-5, C-6 OH-14 1.67 s C-4, C-8, C-14, C-15 3

Figure S1. Effect of compounds 1 and 2 on N-type (Ca v 2.2), T-type (Ca v 3.1), and L-type (Ca v 1.2) calcium channels. A and B. Normalized current-voltage (I-V) curves of Ca v 2.2 in the absence and presence of 30 M 1 and 2, respectively; C and D. Normalized current-voltage (I-V) curves of Ca v 3.1 in the absence and presence of 30 M 1 and 2, respectively; E and F. Normalized current-voltage (I-V) curves of Ca v 1.2 in the absence and presence of 30 M 1 and 2, respectively. Data points reflect mean ± SEM of three determinations. Figure S2. Effect of compounds 1 and 2 on herg. A and B. Normalized current-voltage (I-V) curves of herg in the absence and presence of 30 M 1 and 2, respectively; Data points reflect mean ± SEM of three determinations. 4

Experimental section General experimental procedures UV spectra was obtained using Shimadzu UV2401PC spectrophotometry. Optical rotations were recorded with a Jasco P-1020 polarimeter. 1 H and 13 C NMR spectra were measured on Bruker AV-400 and DRX-500 instruments (Bruker, Zurich, Switzerland) using TMS as internal standard for chemical shifts. Chemical shifts ( ) were expressed in ppm with reference to solvent peak ( H 7.26 and C 77.0). ESIMS and HRTOF-ESIMS data were recorded on API QSTAR Pulsar spectrometer. EIMS and HRTOF-EIMS data were taken on an Waters Auto Spec Premierp 776 spectrometer (America, Waters). Infrared spectra were recorded on a Bruker Tensor-27 instrument by using KBr pellets. An agilent 1100 series instrument equipped with Agilent ZORBAX SB-C18 column (5 μm, 4.6 mm 250 mm) was used for high-performance liquid chromatography (HPLC) analysis. TLC was performed on precoated TLC plates (F254 Si gel 60, Qingdao Marine Chemical, Inc.) with compounds visualized by spraying the dried plates with 5% aqueous H 2 SO 4 followed by heating until dryness. Silica gel (200-300) mesh, Qingdao Marine Chemical, Inc.), Lichroprep RP-18 (40-63 μm, Fuji) and Sephadex LH-20 (20-150 μm, Pharmacia) were used for column chromatography. Methanol, chloroform, ethyl acetate, acetone, petroleum ether and 2-propanol were purchased from Tianjing Chemical Reagents Co. (Tianjing, China). All other materials were of the highest grade available. Plant material The whole plants of Euphorbia peplus were collected from September of 2013 on Kunming, P. R. China, and identified by Prof. M-H Qiu, Kunming Institute of Botany (KIB), Chinese Academy of Sciences. A voucher specimen has been deposited in the herbarium of KIB (number kep-09-13). Extraction and isolation The air-dried powder of the plant (25 kg) was extracted twice with acetone (100 L), heat and reflux for 4 hours, to give 6 kg of crude extract which was suspended in water (20L) and partitioned with petroleum ether (PE) (5 20L). The PE-soluble part (3 kg) was fractioned by a macroporous resin (AB-8) column eluted with MeOH/H 2 O (50% 100%) to give four fractions F1 F4. Fraction F2 (430 g) was extensively subjected to silica gel to give eight subfractions (subf1 F8). subf2 (20 g) were further subjected to RP-18 silica gel, eluted with MeOH:H 2 O (50:50 to 90:10), to obtain 71 fractions. subf2-22 (40 mg) was further purified by semi-preparative HPLC (Acetonile:Water = 70:30, 3 ml/min, wavelength at 210 nm) to afford 2 (6.4 mg, T R = 14.2 min). subf2-45 (120 mg) was chromatographed on LH20 (eluted with 100% MeOH) to yield four fractions (subf2-45a to subf2 45d). subf2-45b was purified by semi-preparative HPLC (Acetonile:Water = 75:25, 3 ml/min, wavelength at 210 nm) to afford 1 (9.5 mg, T R = 15.3 min). 5

Physical constants and spectral data of 1 2 Pepluanol C (1): Colorless needle crystals; [α] 25 D +41.1 (0.25, MeOH); UV (MeOH) λ max (log ε) 210 (4.37) nm; IR (KBr) υ max 3443, 2961, 2928, 1729, 1713, 1645, 1458, 1379, 1234, 1151, 1087, 1042, 996, 942 cm -1 ; 1 H and 13 C NMR data (see table S1); (+)HRESIMS m/z [M + Na] + 437.2309 (calcd for C 25 H 34 O 5 Na, 437.2304). Pepluanol D (2): Colorless needle crystals; [α] 25 D -54.5 (0.25, MeOH); UV (MeOH) λ max (log ε) 202 (3.83), 244 (3.86) nm; IR (KBr) υ max 3520, 3463, 2925, 1672, 1456, 1378, 1258, 1090, 1036, 896 cm -1 ; 1 H and 13 C NMR data (see table S1); (+)HRESIMS m/z [M + Na] + 355.1885 (calcd for C 20 H 28 O 4 Na, 355.1885). Bioassay methods 1. Oocyte preparation and expression Oocytes were obtained from adult Xenopus laevis by a surgical procedure approved by Kunming Institute of Zoology. Preparation of mrna and its expression in oocytes were performed following ref (PMC2862575). 2. Electrophysiology All experiments were performed at 20-22 o C. Whole-oocyte recordings were conducted by two-electrode voltage clamp. Electrodes were filled with KCl (3mM) and had resistances of 0.3-1 MΩ. The bath solution used to record K v 1.3 and herg currents contained NaCl (96 mm), KCl (2.0 mm), MgCl 2 (1mM), Hepes (5 mm),cacl 2 (1.8 mm). ph was adjusted to 7.4 with NaOH and the solution was filtered. All the calcium channels were recorded in Ba 2+ solution (NaOH 50 mm, KCl 2 mm, HEPES 5 mm, BaCl 2 1.8 mm, Ba(OH) 2 40 mm, PH adjusted to 7.4 with Methanesulfonic acid). Kv1.3 currents were evoked from a holding potential of -80 mv by 200-ms depolarizations ranging from -40 mv to +60 mv in 10-mV increments at a 15-s interval; Ca v 2.2 currents were evoked from a holding potential (HP) of -80 mv by 50 ms depolarization from -30 mv to + 70 mv at 3 s intervals; Ca v 3.1 currents were evoked from a holding potential (HP) of -80 mv by 50 ms depolarization from -50 mv to + 60 mv at 3 s intervals; Ca v 1.2 currents were evoked from a holding potential (HP) of -80 mv by 50 ms depolarization from -30 mv to + 70 mv at 3 s intervals; herg were pretreated by indicated compounds for 1 s and then currents were evoked from a holding potential (HP) of -80 mv by 3 s depolarization from -120 mv to + 40 mv at 30 s intervals. All currents were sampled at 10-20 khz and filtered at 5-10 khz. 6

X-ray crystallographic data and deposition information for pepluanol C (1) and D (2) The crystallographic data for 1 (deposition NO. CCDC 1441343) and 2 (deposition NO. CCDC 1441347) have been deposited in Cambridge Crystallographic Data Centre. Copies of the data can be obtained free of charge from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK (Fax: +44-1223-336-033; or E-Mail: deposit@ccdc.cam.ac.uk). X-ray crystallographic data for Pepluanol C (1) Empirical formula C 101 H 138 Cl 2 O 20 [4(C 25 H 34 O 5 ) CH 2 Cl 2 ] Formula weight Temperature Wavelength Crystal system, space group Unit cell dimensions 1743.01 100(2) K 1.54178 A Monoclinic, C 2 a = 28.8519(6) A alpha = 90 deg. b = 12.9422(3) A beta = 113.7510(10) deg. c = 14.1723(3) A gamma = 90 deg. Volume Z, Calculated density Absorption coefficient F(000) 4843.82(18) A^3 2, 1.195 Mg/m^3 1.145 mm^-1 1876 Crystal size Theta range for data collection Limiting indices Reflections collected / unique Completeness to theta = 69.74 Absorption correction Max. and min. transmission Refinement method Data / restraints / parameters Goodness-of-fit on F^2 Final R indices [I>2sigma(I)] R indices (all data) Absolute structure parameter Largest diff. peak and hole 0.72 x 0.13 x 0.11 mm 3.35 to 69.74 deg. -30<=h<=33, -15<=k<=15, -16<=l<=16 23356 / 8051 [R(int) = 0.0496] 93.1 % Semi-empirical from equivalents 0.8844 and 0.4928 Full-matrix least-squares on F^2 8051 / 7 / 573 1.030 R1 = 0.0615, wr2 = 0.1628 R1 = 0.0623, wr2 = 0.1638-0.03(3) 1.896 and -1.131 e.a^-3 View of the hydrogen-bonded motif of 1 Hydrogen-bonds are shown as dashed lines. 7

X-ray crystallographic data for Pepluanol D (2) Empirical formula C 20 H 30 O 5 (C 20 H 28 O 4 H 2 O) Formula weight Temperature Wavelength 350.44 100(2) K 1.54178 A Crystal system, space group Unit cell dimensions Monoclinic, P 21 a = 12.3394(4) A alpha = 90 deg. b = 10.5517(4) A beta = 95.459(2) deg. c = 14.5140(5) A gamma = 90 deg. Volume Z, Calculated density Absorption coefficient F(000) Crystal size Theta range for data collection 1881.18(11) A^3 4, 1.237 Mg/m^3 0.710 mm^-1 760 1.02 x 0.07 x 0.03 mm 3.06 to 70.08 deg. Limiting indices Reflections collected / unique Completeness to theta = 70.08 Absorption correction Max. and min. transmission Refinement method Data / restraints / parameters Goodness-of-fit on F^2 Final R indices [I>2sigma(I)] R indices (all data) Absolute structure parameter Largest diff. peak and hole -14<=h<=15, -11<=k<=10, -16<=l<=17 14672 / 5455 [R(int) = 0.1140] 92.9 % Semi-empirical from equivalents 0.9790 and 0.5313 Full-matrix least-squares on F^2 5455 / 1 / 467 1.132 R1 = 0.0890, wr2 = 0.2300 R1 = 0.1295, wr2 = 0.2587-0.5(4) 1.518 and -0.495 e.a^-3 View of the hydrogen-bonded motif of 2 Hydrogen-bonds are shown as dashed lines. 8

Figure S3: 1 H NMR spectrum of Pepluanol C (1) in CDCl 3 9

Figure S4: 13 C NMR and DEPT spectra of Pepluanol C (1) in CDCl 3 10

Figure S5: HSQC spectrum of Pepluanol C (1) in CDCl 3 11

Figure S6: HMBC spectrum of Pepluanol C (1) in CDCl 3 12

Figure S7: 1 H- 1 H COSY spectrum of Pepluanol C (1) in CDCl 3 13

Figure S8: ROESY spectrum of Pepluanol C (1) in CDCl 3 14

Figure S9: Specific ROESY correlations from OH-4 (δ H 3.26) to H-13 (2.53) of Pepluanol C (1) in CDCl 3 15

Figure S10: HR-ESI (+) MS spectrum of Pepluanol C (1) 16

Figure S11: CD spectrum of Pepluanol C (1) in MeOH File: CD compound 1-1mm(195-400)15102412.dsx ProBinaryX Attributes : - Time Stamp :Sat Oct 24 19:31:27 2015 - File ID : {FA235231-0DC6-49c7-BD9A-2048D615A266} - Is CFR Compliant : false - Original unaltered data Remarks: - HV (CDDC channel): 0 v - Time per point: 1 s - Description: Sample 1 - Concentration: 0.2080mg/mL MeOH - Pathlength: 1 mm Settings: - Time-per-point: 1s (25us x 40000) - Wavelength: 195nm - 400nm - Step Size: 1nm - Bandwidth: 2nm 17

Figure S12: 1 H NMR spectrum of Pepluanol D (2) in CDCl 3 18

Figure S13: 13 C NMR and DEPT spectra of Pepluanol D (2) in CDCl 3 19

Figure S14: HSQC spectrum of Pepluanol D (2) in CDCl 3 20

Figure S15: HMBC spectrum of Pepluanol D (2) in CDCl 3 21

Figure S16: 1 H- 1 H spectrum of Pepluanol D (2) in CDCl 3 22

Figure S17: ROESY spectrum of Pepluanol D (2) in CDCl 3 23

Figure S18: HR-ESI (+) MS spectrum of Pepluanol D (2) 24

Figure S19: CD spectrum of Pepluanol D (2) in MeOH File: CD compound 2-1mm(195-400)15071917.dsx ProBinaryX Attributes : - Time Stamp :Sun Jul 19 16:58:37 2015 - File ID : {86783700-AFD0-4f06-803E-AF873D028853} - Is CFR Compliant : false - Original unaltered data Remarks: - HV (CDDC channel): 0 v - Time per point: 1 s - Description: Sample 1 - Concentration: 0.1300mg/mL MeOH - Pathlength: 1 mm Settings: - Time-per-point: 1s (25us x 40000) - Wavelength: 195nm - 400nm - Step Size: 1nm - Bandwidth: 2nm 25