Supporting Information for

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1 Supporting Information for Cascarinoids A C, A Class of Diterpenoid Alkaloids with Unpredicted Conformations from Croton cascarilloides Xin-Hua Gao,,, Yan-Sheng Xu,, Yao-Yue Fan, Li-She Gan, Jian-Ping Zuo, *,, and Jian-Min Yue *, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai , People s Republic of China University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing , People s Republic of China Laboratory of Immunology and Virology, Shanghai University of Traditional Chinese Medicine, Shanghai , People s Republic of China Institute of Modern Chinese Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou , People s Republic of China X.-H. Gao and Y.-S. Xu contributed equally. List for Supportting Information General Experimental Procedures... 1 Plant Material... 1 Extraction and Isolation... 1 Physical constants and spectral data of X-ray crystallographic analysis D PES scan for croazonoid A (1):... 3 Scheme S1. Biosynthetic Pathway Proposed for Compounds Bioassay, Immunosuppressive Activities Assay... 5 Figure S1. The Cytotoxicity and Immunosuppressive Activities Assay of Compounds Figure S2. Selected COSY and HMBC Correlations for 2 and Table S1. 1 H and 13 C NMR Data in C 5 D 5 N for Table S2. X-ray crystallographic data for cascarinoid A (1) Table S3. X-ray crystallographic data for cascarinoid B (2) Figure S3. 1 H NMR spectrum of cascarinoid A (1) in C 5 D 5 N...12 Figure S4. 13 C NMR spectrum of cascarinoid A (1) in C 5 D 5 N Figure S5. HSQC spectrum of cascarinoid A (1) in C 5 D 5 N Figure S6. 1 H- 1 H COSY spectrum of cascarinoid A (1) in C 5 D 5 N Figure S7. HMBC spectrum of cascarinoid A (1) in C 5 D 5 N Figure S8. NOESY spectrum of cascarinoid A (1) in C 5 D 5 N Figure S9. ESI(+)MS spectrum of cascarinoid A (1)...18 I

2 Figure S10. ESI(-)MS spectrum of cascarinoid A (1)...19 Figure S11. HRESI(+)MS spectrum of cascarinoid A (1)...20 Figure S12. IR spectrum of cascarinoid A (1)...21 Figure S13. 1 H NMR spectrum of cascarinoid B (2) in C 5 D 5 N Figure S C NMR spectrum of cascarinoid B (2) in C 5 D 5 N Figure S15. HSQC spectrum of cascarinoid B (2) in C 5 D 5 N Figure S16. 1 H -1 H COSY spectrum of cascarinoid B (2) in C 5 D 5 N Figure S17. HMBC spectrum of cascarinoid B (2) in C 5 D 5 N Figure S18. NOESY spectrum of cascarinoid B (2) in C 5 D 5 N Figure S19. 1 H spectrum of cascarinoid B (2) in CDCl Figure S C spectrum of cascarinoid B (2) in CDCl Figure S21. HSQC spectrum of cascarinoid B (2) in CDCl Figure S22. 1 H -1 H COSY spectrum of cascarinoid B (2) in CDCl Figure S23. HMBC spectrum of cascarinoid B (2) in CDCl Figure S24. NOESY spectrum of cascarinoid B (2) in CDCl Figure S25. ESI(+)MS spectrum of cascarinoid B (2)...34 Figure S26. ESI(-)MS spectrum of cascarinoid B (2)...35 Figure S27. HRESI(+)MS spectrum of cascarinoid B (2)...36 Figure S28. IR spectrum of cascarinoid B (2)...37 Figure S29. 1 H NMR spectrum of cascarinoid C (3) in C 5 D 5 N Figure S C NMR spectrum of cascarinoid C (3) in C 5 D 5 N Figure S31. HSQC spectrum of cascarinoid C (3) in C 5 D 5 N Figure S32. 1 H- 1 H COSY spectrum of cascarinoid C (3) in C 5 D 5 N Figure S33. HMBC spectrum of cascarinoid C (3) in C 5 D 5 N Figure S34. NOESY spectrum of cascarinoid C (3) in C 5 D 5 N Figure S35. 1 H spectrum of cascarinoid C (3) in CDCl Figure S C spectrum of cascarinoid C (3) in CDCl Figure S37. HSQC spectrum of cascarinoid C (3) in CDCl Figure S38. 1 H- 1 H COSY spectrum of cascarinoid C (3) in CDCl Figure S39. HMBC spectrum of cascarinoid C (3) in CDCl Figure S40. NOESY spectrum of cascarinoid C (3) in CDCl Figure S41. ESI(+)MS spectrum of cascarinoid C (3)...50 Figure S42. ESI(-)MS spectrum of cascarinoid C (3)...51 Figure S43. HRESI(+)MS spectrum of cascarinoid C (3)...52 Figure S44. IR spectrum of cascarinoid C (3)...53 II

3 General Experimental Procedures Optical rotations were determined on an Autopol VI polarimeter. UV data were obtained using a Shimadzu UV-2550 spectrophotometer. IR spectra were acquired on a Thermo IS5 spectrometer with KBr disks. NMR spectra were obtained on a Bruker AM-500 NMR spectrometer. ESIMS and HRESIMS were obtained on a Bruker Daltonics Esquire 3000 plus and a Waters-Micromass Q-TQF Ultima Global mass spectrometer, respectively. Semipreparative HPLC was performed on a Waters 1525 binary pump system with a Waters 2489 detector (210 nm) using a YMC-Pack ODS-A ( mm, S-5 μm). Silica gel ( mesh, Qingdao Haiyang Chemical Co., Ltd.), C18 reversed-phase (RP-C18) silica gel (20 45 μm, Fuji Silysia Chemical Ltd.), CHP20P MCI gel ( μm, Mitsubishi Chemical Corporation), D101-macroporous absorption resin (Shanghai Hualing Resin Co., Ltd.), and Sephadex LH-20 gel (Amersham Biosciences) were used for column chromatography (CC). Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd.) were used for TLC monitors. All solvents used for CC were of analytical grade (Shanghai Chemical Reagents Co., Ltd.), and solvents used for HPLC were of HPLC grade (J & K Scientific Ltd.) Plant Material The twigs and leaves of C. cascarilloides were collected from Xishuangbanna in Yunnan Province, People s Republic of China, and were authenticated by Prof. Y.-K. Xu of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences (CAS). A voucher specimen (accession no. CC-2014GX-1Y) has been deposited in the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Extraction and Isolation Dried powder of C. cascarilloides (3.6 kg) was extracted with 95% EtOH at room temperature to give a crude extract (200 g), which was then partitioned between 1

4 EtOAc and H 2 O. The EtOAc soluble fraction (90 g) was subjected to CC (D101-macroporous absorption resin) eluted with 30%, 50%, 80% and 95% MeOH in H 2 O to give four fractions 1 4, respectively. Fraction 3 (37 g) was separated by an MCI gel column (MeOH/H 2 O, 5:5 to 10:0) to afford three fractions A F. Fraction C (10 g) was chromatographed over a silica gel column and eluted with petroleum ether-acetone (from 50:1 to 1:5) in gradient to afford ten subfractions A1 A10. Fraction A9 (523 mg) was purified by silica gel CC (CH 2 Cl 2 -MeOH, 250:1 to 50:1) and then semipreparative HPLC (40% CH 3 CN in H 2 O, 3 ml/min) to yield compounds 1 (3.5 mg), 2 (6.8 mg), and 3 (8.6 mg), respectively. Physical constants and spectral data of 1 3 Cascarinoid A (1): colorless crystals; mp C; [α] 22.2 D (c 0.321, MeOH); IR (KBr): ν max 3431, 2923, 1745, 1653, 1641, 1617, 1065 cm 1 ; UV/Vis (EtOH): λ max (log ε) = 218 (4.46), 278 (3.51) nm; CD (MeOH): λ (Δε) 207 ( 19.2), 306 (+5.7) nm; 1 H and 13 C data in pyridine-d 5 see Table S1; (±)-ESIMS: m/z [M + H] +, [2 M + Na] +, [M + HCOO], [2 M H] ; (+)-HRESIMS: m/z [M + H] + (calcd for C 28 H 32 NO 5, ). Cascarinoid B (2): colorless crystals; mp C; [α] 22.2 D +2.8 (c 0.107, MeOH); IR (KBr): ν max 3431, 2923, 2853, 1744, 1654, 1641, 1617 cm 1 ; UV/Vis (EtOH): λ max (log ε) = 218 (4.32), 277 (3.50) nm; CD (MeOH): λ (Δε) 200 ( 3.0), 205 ( 2.2), 216 ( 4.4), 234 (2.1), 311 ( 1.2)nm; 1 H and 13 C data in pyridine-d 5 see Table S1 and in CDCl 3 : 207.4, 37.9, 31.0, 63.9, 55.7, 62.4, 47.5, 151.3, 60.3, 36.0, 33.8, 143.6, 44.0, 67.9, 133.5, 170.6, 10.5, 114.9, 14.3, 18.8, 41.4, 34.0, 130.2, 129.8, 115.6, 155.1, 115.6, 129.8ppm; (±)-ESIMS: m/z [M + H] +, [2 M + Na] +, [2M H] ; (+)-HRESIMS: m/z [M + H] + (calcd for C 28 H 32 NO 5, ). Cascarinoid C (3): white powder; [α] 22.2 D (c 0.290, MeOH); IR (KBr): ν max 3431, 2926, 1745, 1676, 1653, 1616 cm 1 ; UV/Vis (EtOH): λ max (log ε) = 218 (4.32), 273 (3.54) nm; CD (MeOH): λ (Δε) 196 ( 3.5), 201 ( 3.0), 213 ( 7.6), 256 (1.4), 315 ( 0.8)nm; 1 H and 13 C data in pyridine-d 5 see Table S1 and in CDCl 3 : 206.9, 38.2, 31.1, 64.8, 56.2, 61.9, 49.2, 152.2, 92.1, 42.0, 34.7, 144.7, 41.7, 67.1, 133.8, 2

5 169.2, 9.8, 115.1, 15.2, 20.4, 41.4, 33.5, 130.9, 130.2, 115.8, 155.1, 115.8, 130.2ppm; (±)-ESIMS: m/z [M + H] +, [2 M + Na] +, [M + HCOO], [2 M H] ; (+)-HRESIMS: m/z [M + H] + (calcd for C 28 H 32 NO 6, ). X-ray crystallographic analysis Cascarinoids A (1) and B (2) were crystallized from MeOH and MeCN at room temperature, respectively. The X-ray crystallographic data for 1 and 2 were obtained on a Bruker SMART CCD detector employing graphite monochromated Cu Kα ϕ-ω scanmode). The structure were solved by direct method using SHELXS-97 (Sheldrick 2008) and refined with full-matrix least-squares calculations on F2 using SHELXL-97 (Sheldrick2008). All non-hydrogen atoms were refined anisotropically. The hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms. Crystallographic data for cascarinoids A (1) and B (2) have been deposited at the Cambridge Crystallographic Data Center with the deposition number of CCDC for 1 and CCDC for 2, respectively. A copy of the data can be obtained free of charge on application to the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [tel: (+44) ; fax: (+44) ; deposit0@ccdc.cam.ac.uk] 1D PES scan for croazonoid A (1): Firstly, the lowest energy conformation of croazonoid A (1) was identified from a Monte Carlo conformational searching using molecular mechanism with MMFF94 force field in the Spartan 08 program. 1 The resulted lowest energy conformer showed similar geometry as the single crystal X-ray diffraction resulted one, and was then subjected to 1D PES scan on the dihedral angle of C(3')-C(2')-C(1')-N(1). Secondly, relaxed PES scans were performed with and without dispersion correction at the HF/STO-3G and B3LYP/6-31G level in Gaussian 09 software package, 2 respectively. For each scan, the internal redundant coordinates and 36 steps of 10 degree increasing 3

6 in the dihedral angle was applied. Finally, to better understanding and comparing of the results, the relative energy of the extended lowest energy conformer in each scan, whose dihedral angle of C(3')-C(2')-C(1')-N(1) near 180 degree, was set as 0 Kcal/mol. References 1. Spartan 08; Wavefunction Inc.:Irvine, CA. 2. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, Jr., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09 Revision D. 01, Gaussian, Inc., Wallingford CT, Scheme S1. Biosynthetic Pathway Proposed for Compounds 1 3. The biosynthetic pathway for compounds 1 3 was proposed (Scheme S1) with geranylgeranyl pyrophosphate (GGPP) as the precursor, which was first transformed 4

7 into crotofolane-type diterpenoid skeleton as the key intermediate ii. The intermediate ii subsequently underwent aminolysis 1 and oxidative modifications to produce compounds 1 3, respectively. References 1. Wang, F. P.; Liang, X. T. C20-diterpenoid alkaloids. In Alkaloids Chem. Biol., Academic Press: 2002, 59, Bioassay, Immunosuppressive Activities Assay Reagents: Concanavalin A (Con A), lipopolysaccharide (LPS, Escherichia coli 055:B5), CCK-8: WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt], and RPMI 1640 medium were purchased from GibcoBRL, Life Technologies (USA). Fetal bovine serum (FBS) was purchased from HyClone Laboratories (Utah, USA). [ 3 H]-Thymidine (10μ Ci ml 1 ) was obtained from the Shanghai Institute of Atomic Energy (SIAE). Animals: Female BALB/c mice (18 20g) were obtained from Shanghai Experimental Animal Center and were housed in specific conditions (12 h light/12 h dark photoperiod, 22 1 C, 55% ± 5% relative humidity). All husbandry and experimental contact were made with the mice maintained specific pathogen-free conditions. All experiments were carried out according to the NIH Guidelines for Care and Use of Laboratory Animals and approved by the Bioethics Committee of Shanghai Institute of Materia Medica. Preparation of spleen cells from mice: Female BALB/c mice were sacrificed by cervical dislocation, and the spleens were removed aseptically. Mononuclear cell suspensions were prepared after cell debris, and clumps were removed. Erythrocytes were depleted with ammonium chloride buffer solution. Lymphocytes were washed and resuspended in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U ml 1 ), and streptomycin (100 mg ml 1 ). Cytotoxicity assay: Cytotoxicity was assessed with CCK-8 assay. Briefly, fresh spleen cells were obtained from female BALB/c mice (18 20g). Spleen cells ( cells) were cultured at 37 C for 48 h in 96-well flat plates, in the presence or absence 5

8 of various concentrations of compounds, in a humidified and 5% CO 2 -containing incubator. A certain amount of CCK-8 was added to each well at the final 8 10 h of culture. To the end of the culture, we measured the OD values with microplate reader (Bio-Rad 650) at 450 nm. The cytotoxicity of each compound was expressed as the concentration of compound that reduced cell viability to 50% (CC 50 ). T cell and B cell function assay: Fresh spleen cells were obtained from female BALB/c mice (18 20g). The spleen cells were cultured at the same conditions as those mentioned above. The cultures, in the presence or absence of various concentrations of compounds, were stimulated with 5 μg ml 1 of ConA or 10 μg ml 1 of LPS to induce T cells or B cells proliferative responses, respectively. Proliferation was assessed in terms of uptake of [ 3 H]-thymidine during 8 h of pulsing with 25μ L/well of [ 3 H]-thymidine, and then cells will be harvested onto glass fiber filters. The incorporated radioactivity was counted using a Beta scintillation counter (MicroBeta Trilux, PerkinElmer Life Sciences). The immunosuppressive activity of each compound was expressed as the concentration of compound that inhibited ConA-induced T cell proliferation or LPS-induced B cell proliferation to 50% (IC 50 ) of the control value. 6

9 Figure S1. The Cytotoxicity and Immunosuppressive Activities Assay of Compounds 2 3. (a and d: cytotoxicity of compounds 2 3 were tested in BALB/c mice splenocytes using the CCK8 assay on a 48-h culture, with CC 50 values of µm and µm; b and e: compounds 2 3 inhibited ConA-induced splenocyte proliferation in a 48-h culture, with IC 50 values of >1000 µm and 6.14 µm; c and f: compounds 2 3 inhibited LPS-induced splenocyte proliferation in a 48-h culture, with IC 50 values of µm and µm. The CC 50 and the IC 50 values were counted by using the Graphpad Prism 6.0 statistical software.) 7

10 Figure S2. Selected COSY and HMBC Correlations for 2 and 3 8

11 Table S1. 1 H and 13 C NMR Data in C 5 D 5 N for 1 3. no. δ H, mult (J, Hz) a δ C b δ H, mult (J, Hz) a δ C b δ H, mult (J, Hz) a δ C b , ddd (8.5, 8.3, 7.1) , ddd (8.2, 7.8, 7.2) , ddd (8.3, 7.3, 7.0) , dd (13.6, 8.3) , dd (13.7, 7.8) , dd (14.0, 7.0) , dd (13.6, 8.5 ) 2.61, dd (13.7, 8.2) 2.64, dd (14.0,8.3) , s , s , s , d (12.6) , d (12.8) , d (12.5) , brd (11.5, 3.5) , dd (10.2, 5.0) , ddd (12.7, 12.4, 4.3) , m , m , ddd (12.4, 11.6, 3.9) 2.37, m 2.73, ddd (13.0, 5.1, 4.9) , m , m , m , ddd (13.1, 4.2) 2.41, m 2.64, m , d (12.6) , d (12.8) , d (12.5) , s , brs , brs a 5.18, s , s , s b 4.97, s 5.19, s 5.32, s , d (7.1) , d (7.3) , d (7.3) , s , s , s , ddd (14.1, 7.1, 7.0) , ddd (14.3, 7.9, 6.4) , ddd (13.9, 11.1, 5.3) , ddd (14.1, 7.2, 7.0) 4.33, ddd (14.3, 8.2, 6.5) 3.88, ddd (13.9, 11.1, 5.4) , ddd (14.7, 7.0, 7.0) , ddd (14.3, 7.9, 6.5) , ddd (13.2, 11.1, 5.4) , ddd (14.7, 7.2, 7.1) 3.02, ddd (14.3, 8.2, 6.4) 3.21, ddd (13.2, 11.1, 5.3) , d (8.4) , d (8.3) , d (8.3) , d (8.4) , d (8.3) , d (8.3) , d (8.4) , d (8.3) , d (8.2) , d (8.4) , d (8.3) , d (8.3) OH 7.97, s 6 -OH 11.41, s 11.38, s 11.31, s a recorded at 400 MHz, b recorded at 125 MHz. 9

12 Table S2. X-ray crystallographic data for cascarinoid A (1). Identification code cu_dm14634_0m Empirical formula C28 H31 N O5 Formula weight Temperature 130 K Wavelength Å Crystal system Orthorhombic Space group P Unit cell dimensions a = (2) Å b = (2) Å c = (2) Å Volume (6) Å 3 Z 4Density (calculated) Mg/m 3 Absorption coefficient mm -1 F(000) 984 Crystal size 0.3 x 0.25 x 0.22 mm 3 Theta range for data collection to Index ranges -15<=h<=14, -15<=k<=15, -16<=l<=16 Reflections collected Independent reflections 4260 [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 4260 / 0 / 311 Goodness-of-fit on F Final R indices [I>2sigma(I)] R1 = , wr2 = R indices (all data) R1 = , wr2 = Absolute structure parameter -0.09(5) Extinction coefficient n/a Largest diff. peak and hole and e.å -3 Colorless crystals of 1 was obtained in MeOH. 10

13 Table S3. X-ray crystallographic data for cascarinoid B (2). Identification code cu_dm17215_0m Empirical formula C30 H34 N2 O5 Formula weight Temperature K Wavelength Å Crystal system Monoclinic Space group P Unit cell dimensions a = (2) Å = 90. b = 8.799(2) Å = (14). c = (3) Å = 90. Volume (5) Å 3 Z 2 Density (calculated) Mg/m 3 Absorption coefficient mm -1 F(000) 536 Crystal size 0.15 x 0.12 x 0.08 mm 3 Theta range for data collection to Index ranges -12<=h<=12, -9<=k<=10, -17<=l<=17 Reflections collected Independent reflections 4189 [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 4189 / 1 / 339 Goodness-of-fit on F Final R indices [I>2sigma(I)] R1 = , wr2 = R indices (all data) R1 = , wr2 = Absolute structure parameter -0.4(2) Extinction coefficient n/a Largest diff. peak and hole and e.å -3 Colorless crystals of 2 was obtained in MeCN. 11

14 Figure S3. 1 H NMR spectrum of cascarinoid A (1) in C 5 D 5 N. 12

15 Figure S4. 13 C NMR spectrum of cascarinoid A (1) in C 5 D 5 N. 13

16 Figure S5. HSQC spectrum of cascarinoid A (1) in C 5 D 5 N. 14

17 Figure S6. 1 H- 1 H COSY spectrum of cascarinoid A (1) in C 5 D 5 N. 15

18 Figure S7. HMBC spectrum of cascarinoid A (1) in C 5 D 5 N. 16

19 Figure S8. NOESY spectrum of cascarinoid A (1) in C 5 D 5 N. 17

20 Figure S9. ESI(+)MS spectrum of cascarinoid A (1) 18

21 Figure S10. ESI(-)MS spectrum of cascarinoid A (1) 19

22 Figure S11. HRESI(+)MS spectrum of cascarinoid A (1) 20

23 Figure S12. IR spectrum of cascarinoid A (1) 21

24 Figure S13. 1 H NMR spectrum of cascarinoid B (2) in C 5 D 5 N. 22

25 Figure S C NMR spectrum of cascarinoid B (2) in C 5 D 5 N. 23

26 Figure S15. HSQC spectrum of cascarinoid B (2) in C 5 D 5 N. 24

27 Figure S16. 1 H -1 H COSY spectrum of cascarinoid B (2) in C 5 D 5 N. 25

28 Figure S17. HMBC spectrum of cascarinoid B (2) in C 5 D 5 N. 26

29 Figure S18. NOESY spectrum of cascarinoid B (2) in C 5 D 5 N. 27

30 Figure S19. 1 H spectrum of cascarinoid B (2) in CDCl3 28

31 Figure S C spectrum of cascarinoid B (2) in CDCl 3 29

32 Figure S21. HSQC spectrum of cascarinoid B (2) in CDCl 3 30

33 Figure S22. 1 H -1 H COSY spectrum of cascarinoid B (2) in CDCl 3 31

34 Figure S23. HMBC spectrum of cascarinoid B (2) in CDCl 3 32

35 Figure S24. NOESY spectrum of cascarinoid B (2) in CDCl 3 33

36 Figure S25. ESI(+)MS spectrum of cascarinoid B (2) 34

37 Figure S26. ESI(-)MS spectrum of cascarinoid B (2) 35

38 Figure S27. HRESI(+)MS spectrum of cascarinoid B (2) 36

39 Figure S28. IR spectrum of cascarinoid B (2) 37

40 Figure S29. 1 H NMR spectrum of cascarinoid C (3) in C 5 D 5 N. 38

41 Figure S C NMR spectrum of cascarinoid C (3) in C 5 D 5 N. 39

42 Figure S31. HSQC spectrum of cascarinoid C (3) in C 5 D 5 N. 40

43 Figure S32. 1 H- 1 H COSY spectrum of cascarinoid C (3) in C 5 D 5 N. 41

44 Figure S33. HMBC spectrum of cascarinoid C (3) in C 5 D 5 N. 42

45 Figure S34. NOESY spectrum of cascarinoid C (3) in C 5 D 5 N. 43

46 Figure S35. 1 H spectrum of cascarinoid C (3) in CDCl 3 44

47 Figure S C spectrum of cascarinoid C (3) in CDCl 3 45

48 Figure S37. HSQC spectrum of cascarinoid C (3) in CDCl 3 46

49 Figure S38. 1 H- 1 H COSY spectrum of cascarinoid C (3) in CDCl 3 47

50 Figure S39. HMBC spectrum of cascarinoid C (3) in CDCl 3 48

51 Figure S40. NOESY spectrum of cascarinoid C (3) in CDCl 3 49

52 Figure S41. ESI(+)MS spectrum of cascarinoid C (3) 50

53 Figure S42. ESI(-)MS spectrum of cascarinoid C (3) 51

54 Figure S43. HRESI(+)MS spectrum of cascarinoid C (3) 52

55 Figure S44. IR spectrum of cascarinoid C (3) 53