Kadcoccinones A F, New Biogenetically Related Lanostane-Type. Triterpenoids with Diverse Skeletons from Kadsura coccinea

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1 Kadcoccinones A F, New Biogenetically Related Lanostane-Type Triterpenoids with Diverse Skeletons from Kadsura coccinea Zheng-Xi Hu,,, Yi-Ming Shi,, Wei-Guang Wang, Xiao-Nian Li, Xue Du, Miao Liu, Yan Li, Yong-Bo Xue, Yong-Hui Zhang, *, Jian-Xin Pu, *, and Han-Dong Sun State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming , China Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan , China * Corresponding author Tel.: (86) zhangyh@mails.tjmu.edu.cn; pujianxin@mail.kib.ac.cn. 1

2 Contents of Supporting Information Contents Pages Detailed experimental procedures and physical-chemical properties H and 13 C NMR assignments of compounds Calculated ECD spectra of compound 3 11 Selected HMBC and 1 H- 1 H COSY correlations of compounds 1, 2 and Selected ROESY correlations of compound 2 12 Selected ROESY correlations of compound 3 12 X-ray crystallographic structures of compounds 1 and D and 2D NMR spectra of compounds HRESIMS, ESI, IR, UV, and ECD spectra of compounds Computational data of compound References 93 2

3 Detailed experimental procedures 1. General Experimental Procedures Optical rotations were recorded on a JASCO P-1020 digital polarimeter. UV spectra were measured on a Shimadzu UV2401PC spectrophotometer. Experimental ECD spectra were obtained on a Chirascan instrument. IR spectra were measured on a Tenor 27 FT-IR spectrometer with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker AM-400 and DRX-600 spectrometers using TMS as the internal standard. All chemical shifts (δ) were expressed in ppm relative to the solvent signals. HRESIMS were performed on an API QSTAR Pulsar 1 spectrometer. X-ray crystallographic data were obtained on a Bruker APEX DUO diffractometer equipped with an APEX II CCD using Cu Kα radiation. Column chromatography was performed with silica gel ( mesh and mesh, Qingdao Marine Chemical, Inc., Qingdao, People s Republic of China), and Lichroprep RP-18 gel (40 63 μm, Merck, Darmstadt, Germany). Thin-layer chromatography was performed on precoated TLC plates ( μm thickness, silica gel 60 F 254, Qingdao Marine Chemical, Inc.). Fractions were monitored by TLC and spots were visualized by spraying heated silica gel plates with 10% H 2 SO 4 in EtOH. Preparative HPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph with a Shimadzu PRC ODS column. Semipreparative HPLC was performed on an Agilent 1200 liquid chromatograph with Zorbax SB-C 18 (9.4 mm 250 mm) and Zorbax Rx-SIL (9.4 mm 250 mm) columns. 2. Plant Material The stems of K. coccinea were collected in the Menglun District of Mengla County, Yunnan Province, People s Republic of China, in September 2009 and identified by Prof. Xi-Wen Li at the Kunming Institute of Botany. A voucher specimen (KIB ) has been deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences. 3. Extraction, isolation, and purification of compounds 1 6 The air-dried and powdered stems (9.0 kg) were extracted with 70% aqueous acetone (45 L) three times (three days each time) at room temperature. After removal of the solvent under reduced pressure in vacuo, the total extract was suspended in water, and then extracted 3

4 exhaustively with EtOAc and n-buoh, respectively. The EtOAc residue (450.0 g) was subjected to silica gel column with a CHCl 3 /CH 3 OH gradient system (1:0 to 0:1) to afford six fractions A F. Fraction B (150.0 g) was chromatographed by RP-C 18 silica gel column with a step-wise gradient of MeOH/H 2 O (30:70 to 100:0) to obtain fractions B1 B7. Fraction B6 (40.0 g) was subjected to silica gel column with a gradient elution of MeOH/H 2 O (65:35 to 100:0) to obtain nine fractions (B6.1 B6.9). Fraction B6.8 (25.0 g) was fractioned by silica gel column eluted with petroleum ether/me 2 CO (5:1 to 2:1) to give eight fractions (B6.8a B6.8h). Fraction B6.8c (2.0 g) was subjected to repeated semipreparative HPLC (MeOH/H 2 O, 90:10) to afford 6 (28.3 mg). Fraction B6.8f (1.6 g) was purified by repeated semipreparative HPLC (MeOH/H 2 O, 85:15) to obtain 3 (5.0 mg). Fraction B7 (50.0 g) was purified by semi-preparative HPLC (MeOH/H 2 O, 92:8) to yield 2 (114.3 mg). B6.7 (6.0 g) was subjected to RP-C 18 silica gel column eluted with MeOH/H 2 O (70:30 to 100:0) to yield ten fractions (B6.7a B6.7j). B6.7h (1.2 g) was chromatographed on silica gel column eluted with petroleum ether/me 2 CO (8:1 to 4:1), and then subjected to preparative HPLC on an Rp-18 column (15 ml/min, MeCN/H 2 O, 80:20 to 95:5 in 20 min) to obtain a mixture, which was subjected to semi-preparative HPLC (MeOH/H 2 O, 85:15) to yield 1 (30.0 mg). B6.7f (1.1 g) was subjected to silica gel column eluted with petroleum ether/me 2 CO (8:1 to 5:1), and then subjected to repeated semi-preparative HPLC (cyclohexane/isopropanol/meoh, 96:3.4:0.6, 3 ml/min) (Figure S1) to yield 4 (8.0 mg) and 5 (7.9 mg). DAD1 A, Sig=254,4 Ref=360,100 (HZX\ D) DAD1 B, Sig=365,16 Ref=360,100 (HZX\ D) DAD1 C, Sig=210,8 Ref=360,100 (HZX\ D) DAD1 D, Sig=230,16 Ref=360,100 (HZX\ D) DAD1 E, Sig=280,16 Ref=360,100 (HZX\ D) mau min Figure S1. HPLC profiles of mixtures containing compounds 4 and ECD Calculation and molecular orbital analysis The theoretical calculations of compound 3 were carried out using Gaussian Conformational analysis was initially performed using Discovery Studio 3.5 Client. The optimized conformation geometries, thermodynamic parameters, and populations of all conformations were 4

5 provided in Figure S76 and Tables S4 S7 in the Supporting Information. The conformers were optimized at B3LYP/6-31G(d) level. Room-temperature equilibrium populations were calculated according to Boltzmann distribution law. 13 C NMR shielding constants of compound 3 were calculated with the GIAO method at MPW1PW91-SCRF/6-31G(d,p) level in DMSO with PCM. The shielding constants so obtained were converted into chemical shifts by referencing to TMS at 0 ppm (δ cal = σ TMS σ cal ), where the σ TMS was the shielding constant of TMS calculated at the same level. The parameters a and b of the linear regression δ cal = aδ exp + b and the correlation coefficient, R 2, were presented. The theoretical calculation of ECD were performed using TDDFT at B3LYP/6-31+G(d,p) level in the gas phase and in methanol with PCM. The ECD spectrum of compound 3 was obtained by weighing the Boltzmann distribution rate of each geometric conformation. The ECD spectrum is simulated by overlapping Gaussian functions for each transition according to where σ represents the width of the band at 1/e height, and ΔE i and R i are the excitation energies and rotational strengths for transition i, respectively. σ = 0.20 ev and R velocity have been used in this work. The orbital information (NBO plot files) was generated by NBO program of Gaussian The predominantly populated conformers were selected for molecular orbital (MO) analysis. NBO plot files were used to generate corresponding Gaussian-type grid file by Multiwfn After that, the isosurface of generated grid date was afforded by VMD software Cytotoxicity Assays Six human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW-480, and HeLa) were used in the cytotoxic assay. All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone) at 37 C in a humidified atmosphere with 5% CO 2. The cytotoxicity assay was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Sigma, St. Louis, MO, USA) assay. 5 Briefly, 100 μl of adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before addition of test compound, both with an initial density of 5

6 cells/ml in 100 μl medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cisplatin and paclitaxel (Sigma) as positive controls. After the incubation, MTS (100 μg) was added to each well and the incubation continued at 37 C for 4 h. The cells were lysed with 100 μl of 20% SDS-50% DMF after removal of 100 μl of medium. The optical density of the lysate was measured at 490 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC 50 value of each compound was calculated by the Reed and Muench s method. 6 Table S1. Cytotoxic Activities of Triterpenoids from K. coccinea against Tumor Cell Lines a compound HL-60 SMMC-7721 A-549 MCF-7 SW-480 HeLa > >40 >40 > > DDP b paclitaxel b <0.008 <0.008 <0.008 <0.008 <0.008 <0.008 a Results are expressed as IC 50 values in μm. Cell lines: HL-60, acute leukemia; SMMC-7721, hepatic cancer; A-549, lung cancer; MCF-7, breast cancer; SW-480, colon cancer; HeLa, cervical cancer. Compounds 1, 3, and 6 were inactive (IC 50 > 40 μm) for all tumor cell lines. b DDP (cis-platin) and paclitaxel were used as positive controls. 6. Physical-chemical properties of compounds 1 6 Kadcoccinone A (1): Colorless needle crystals (EtOH); [α] 25 D : 76.1 (MeOH, c 0.1). UV (MeOH) λmax (log ε): 203 (4.36) nm; ECD (MeOH) λmax (Δε): 218 (+ 7.88) nm; IR (ν max ): 3448, 2958, 2928, 2870, 1714, 1635, 1454, 1434, 1384, 1268, 1247, 1110, 1046, and 1030 cm -1. HRESIMS m/z ([M + Na] +, calcd for ). 1 H and 13 C NMR data, see Table S2 and S3. Kadcoccinone B (2): White amorphous powder; [α] 23 D : (MeOH, c 0.1). UV (MeOH) λmax (log ε): 203 (4.36) nm; IR (ν max ): 3438, 2957, 2930, 2870, 1727, 1705, 1633, 1457, 1379, 1246, 1184, 1048, 1028, 604, and 580 cm -1. HRESIMS m/z ([M H], calcd for ). 1 H and 13 C NMR data, see Table S2 and S3. Kadcoccinone C (3): White amorphous powder; [α] 23 D : 32.9 (MeOH, c 0.2); UV (MeOH) λmax (log ε): 203 (4.05) nm; UV (MeOH) λmax (log ε): 203 (4.36) nm; ECD (MeOH) λmax (Δε): 200 ( ), 212 ( 2.33) nm; IR (ν max ): 3432, 2970, 2928, 1727, 1694, 1639, 1547, 1381, 1246, 1186, 1143, 1055, 1030, and 993 cm -1. HRESIMS m/z ([M + Na] +, calcd for ). 1 H and 13 C NMR data, see Table S2 and S3. 6

7 Kadcoccinone D (4): Colorless needle crystals (MeOH); [α] 25 D : 69.4 (MeOH, c 0.2). UV (MeOH) λmax (log ε): 201 (3.46) nm; ECD (MeOH) λmax (Δε): 211 (+ 0.79), 289 ( 0.58) nm; IR (ν max ): 3571, 2962, 2931, 2865, 1714, 1631, 1446, 1425, 1384, 1251, 1057, 1033, and 879 cm -1. HRESIMS m/z ([M + Na] +, calcd for ). 1 H and 13 C NMR data, see Table S2 and S3. Kadcoccinone E (5): Colorless needle crystals (MeOH); [α] 25 D : (MeOH, c 0.3). UV (MeOH) λmax (log ε): 203 (4.10) nm; ECD (MeOH) λmax (Δε): 211 (+ 2.42), 288 (+ 2.42) nm; IR (ν max ): 3496, 2965, 2929, 2875, 1705, 1630, 1450, 1382, 1363, 1257, 1059, 988, and 881 cm -1. HRESIMS m/z ([M + Na] +, calcd for ). 1 H and 13 C NMR data, see Table S2 and S3. Kadcoccinone F (6): Colorless needle crystals (MeOH); [α] 22 D : (MeOH, c 0.1). UV (MeOH) λmax (log ε): 204 (4.01) nm; IR (ν max ): 2951, 2875, 1730, 1710, 1631, 1551, 1459, 1414, 1381, 1245, 1182, 1049, 1029, 983, and 603 cm -1. HRESIMS m/z ([M H], calcd for ). 1 H and 13 C NMR data, see Table S2 and S3. Crystal data for kadcoccinone A (1): C 32 H 48 O 5 C 2 H 6 O, M = , monoclinic, a = (3) Å, b = (14) Å, c = (5) Å, α = 90.00, β = (2), γ = 90.00, V = (2) Å 3, T = 100(2) K, space group P21, Z = 4, μ(cukα) = mm -1, reflections measured, independent reflections (R int = ). The final R 1 values were (I > 2σ(I)). The final wr(f 2 ) values were (I > 2σ(I)). The final R 1 values were (all data). The final wr(f 2 ) values were (all data). The goodness of fit on F 2 was Flack parameter = 0.1(2). The Hooft parameter is 0.30(10) for 4776 Bijvoet pairs. Crystal data for kadcoccinone D (4): C 29 H 44 O 4, M = , orthorhombic, a = (2) Å, b = (4) Å, c = (11) Å, α = 90.00, β = 90.00, γ = 90.00, V = (15) Å 3, T = 100(2) K, space group P212121, Z = 4, μ(cukα) = mm -1, reflections measured, 4498 independent reflections (R int = ). The final R 1 values were (I > 2σ(I)). The final wr(f 2 ) values were (I > 2σ(I)). The final R 1 values were (all data). The final wr(f 2 ) values were (all data). The goodness of fit on F 2 was Flack parameter = 0.07(18). The Hooft parameter is 0.05(9) for 1808 Bijvoet pairs. Crystal data for kadcoccinone E (5): C 29 H 44 O 4, M = , triclinic, a = (3) Å, b = (5) Å, c = (6) Å, α = (2), β = (2), γ = (10), V = 7

8 (9) Å 3, T = 100(2) K, space group P1, Z = 2, μ(cukα) = mm -1, reflections measured, 5187 independent reflections (R int = ). The final R 1 values were (I > 2σ(I)). The final wr(f 2 ) values were (I > 2σ(I)). The final R 1 values were (all data). The final wr(f 2 ) values were (all data). The goodness of fit on F 2 was Flack parameter = 0.4(3). Crystal data for kadcoccinone F (6): C 32 H 48 O 5, M = , monoclinic, a = (10) Å, b = (2) Å, c = (3) Å, α = 90.00, β = 97.23, γ = 90.00, V = (4) Å 3, T = 100(2) K, space group P21, Z = 2, μ(cukα) = mm -1, reflections measured, 4341 independent reflections (R int = ). The final R 1 values were (I > 2σ(I)). The final wr(f 2 ) values were (I > 2σ(I)). The final R 1 values were (all data). The final wr(f 2 ) values were (all data). The goodness of fit on F 2 was Flack parameter = 0.4(3). The Hooft parameter is 0.01(9) for 1636 Bijvoet pairs. The crystallographic data for 1 (deposition No. CCDC ), 4 (deposition No. CCDC ), 5 (deposition No. CCDC ), and 6 (deposition No. CCDC ) have been deposited in the 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: ; or deposit@ccdc.cam.ac.uk). 8

9 1 H and 13 C NMR assignments of compounds 1 6 Table S2. 1 H NMR Spectroscopic Data for 1 6 (δ in ppm). NO. 1 a,c 2 a,c 3 a,c 3 b,c 4 a,c 5 a,c 6 a,c m; 1.77 m 1.07 m ;1.76 m 0.97 m; 2.46 m 1.02 m; 1.87 m 0.98 m; 0.99 m 2.48 dt (3.5, 13.5) 2.48 dt (3.3, 13.5) 1.16 m; 1.76 m m; 1.72 m 1.64 m; 1.84 m 1.71 m; 1.86 m 1.59 m; 1.91 m 1.78 m; 2.01 m 1.79 m; 2.02 m 1.73 m; 1.86 m br s 4.84 br s 4.83 br s 4.57 br s 3.67 br s 3.67 br s 4.85 br s dd (5.3, 10.6) 1.50 dd (5.6, 10.2) 1.59 dd (5.7, 11.1) 1.51 dd (4.5, 12.1) 1.97 m 1.98 m 1.39 dd (4.6, 10.8) m 1.94 m 1.88 m 1.93 m 1.97 m; 2.06 m 1.97 m; 2.06 m 1.93 m m 5.53 m 5.74 br d (3.4) 5.61 br d (5.5) 5.61 br d (5.8) 5.62 br d (5.8) 5.74 m br t (10.4) 2.48 br t (10.3) 2.46 br d (12.3) 2.17 br d (13.0) 2.41 dd (7.2, 11.3) 2.42 dd (7.3, 11.1) 2.98 m m; 1.77 m 1.64 m; 1.74 m 1.91 m; 1.38 dd (12.3, 14.2) 2.39 dd (14.1, 18.4); 1.56 m; 1.94 m 1.56 m; 1.94 m 2.94 dd (3.6, 12.3) 2.45 dd (3.3, 12.3) 2.72 dd (6.0, 18.4) dd (4.6, 12.5) 2.19 m m 2.31 m m; 1.89 m 1.24 m; 1.82 m 1.20 m 1.17 dd (7.0, 11.5); dd (7.0, 11.5); 1.05 m; 2.02 m 2.34 dt (3.9, 14.0) m 1.60 m 1.63 m; 1.74 m m; 1.99 m 1.61 m; 1.93 m 1.33 m; 2.22 m 1.16 m; 1.96m 1.69 m; 1.86 m 1.71 m; 1.81 m 1.35 m; 2.09 m m 1.96 m 3.13 q (9.3, 18.2) 2.54 m m d (2.6); 4.81 d (2.0); 4.84 d (2.6) 4.84 d (2.0) 5.11 s; 5.53 s 4.81 br s; 5.15 br s 0.88 d (6.8) 0.88 d (6.7) 1.20 m s 1.07 s 0.97 s 0.88 s 1.02 s 1.05 s 1.07 s m 1.93 m 1.79 m 1.53 m 2.19 m 2.24 m 1.46 m d (6.2) 1.13 d (5.9) 1.19 d (6.3) 0.82 m 1.01 d (7.1) 1.01 d (7.1) 1.23 d (6.6) m; 2.27 m 1.26 m; 1.83 m 1.34 m; 1.93 m 1.17 m; 1.64 m 1.53 m; 1.96 m 1.47 m; 1.97 m 1.35 m; 1.73 m td (4.7, 9.5) 2.82 m; 2.94 m 2.82 m; 2.92 m 2.38 m; 2.53 m 3.45 dt (1.9, 5.8) 3.47 m 2.85 m; 2.91 m dd (1.1, 9.5) 6.02 t (7.4) 6.09 t (7.1) 5.87 br s 3.50 d (1.9) 3.50 d (1.5) 6.06 t (6.9) d (1.1) s 2.18 s 1.80 s 2.05 s 2.07 s 2.15 s s 1.14 s 1.51 s 1.19 s 1.11 s 1.11 s 0.89 s s 0.91 s 0.89 s 0.95 s 0.97 s 0.98 s 0.90 s s 0.92 s 0.82 s 0.83 s 1.23 s 1.24 s 0.92 s -OAc 2.10 s 2.11 s 1.96 s 2.03 s a Recorded at 600 MHz in C 5 D 5 N. b Recorded at 600 MHz in DMSO. c m means overlapped or multiplet with other signals. 9

10 Table S3. 13 C NMR Spectroscopic Data for 1 6 (δ in ppm). NO. 1 a 2 b 3 a 3 c 4 a 5 a 6 a CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH 79.1 CH 78.8 CH 77.6 CH 76.0 CH 76.0 CH 78.4 CH C 36.8 C 36.8 C 35.9 C 37.9 C 37.9 C 37.1 C CH 41.1 CH 39.3 CH 38.3 CH 37.7 CH 37.7 CH 43.9 CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH C C C C C C C CH 51.8 CH 44.7 CH 43.5 CH 46.6 CH 46.7 CH 47.9 CH C 35.1 C 35.2 C 34.1 C 35.5 C 35.5 C 36.3 C CH CH CH CH CH CH CH CH 51.3 CH 98.5 C 97.0 C 27.0 CH 27.0 CH C C C C C 79.2 C 78.7 C 59.3 C C 44.5 C 77.8 C 76.4 C 47.3 C 47.2 C 53.7 C CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH 48.8 CH 42.5 CH 40.8 CH 72.4 C 72.3 C 45.8 CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH 31.6 CH 36.8 CH 35.3 CH 32.3 CH 32.6 CH 38.1 CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH 27.4 CH CH CH CH 58.0 CH 28.2 CH CH CH CH CH 61.1 CH 60.9 CH CH C C C C C C CH CH C C C C C 22.2 CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH CH 3 -OAc C C C C C 21.5 CH CH CH CH CH 3 a Recorded at 150 MHz in C 5 D 5 N. b Recorded at 100 MHz in C 5 D 5 N. c Recorded at 150 MHz in DMSO. 10

11 Calculated ECD spectra of compound 3 Figure S2. Experimental (black) and calculated (red) ECD spectra of 3 in MeOH. Figure S3. Selected HMBC and 1 H- 1 H COSY correlations of compounds 1, 2 and

12 Figure S4. Selected ROESY correlations of compound Å 1.99 Å 3.99 Å 2.39 Å Figure S5. Selected ROESY correlations of compound 3 [the HO-12 were β-direction (left) and α-direction (right)] 12

13 Figure S6. View of the molecule of 1 with the atom-labelling scheme (displacement ellipsoids are drawn at the 30% probability level). Figure S7. View of the hydrogen-bonded motif of 1 (hydrogen-bonds are shown as dashed lines). 13

14 Figure S8. View of the molecule of 4 with the atom-labelling scheme (displacement ellipsoids are drawn at the 30% probability level). Figure S9. View of the hydrogen-bonded motif of 4 (hydrogen-bonds are shown as dashed lines). 14

15 Figure S10. View of the molecule of 5 with the atom-labelling scheme (displacement ellipsoids are drawn at the 30% probability level). Figure S11. View of the hydrogen-bonded motif of 5 (hydrogen-bonds are shown as dashed lines). 15

16 Figure S12. View of the molecule of 6 with the atom-labelling scheme (displacement ellipsoids are drawn at the 30% probability level). Figure S13. View of the hydrogen-bonded motif of 6 (hydrogen-bonds are shown as dashed lines). 16

17 Figure S14. 1 H NMR spectrum of kadcoccinone A (1) in C 5 D 5 N. 17

18 Figure S C NMR spectrum of kadcoccinone A (1) in C 5 D 5 N. 18

19 Figure S16. HSQC spectrum of kadcoccinone A (1) in C 5 D 5 N. 19

20 Figure S17. HMBC spectrum of kadcoccinone A (1) in C 5 D 5 N. 20

21 Figure S18. 1 H 1 H COSY spectrum of kadcoccinone A (1) in C 5 D 5 N. 21

22 Figure S19. ROESY spectrum of kadcoccinone A (1) in C 5 D 5 N. 22

23 Figure S20. 1 H NMR spectrum of kadcoccinone B (2) in C 5 D 5 N. 23

24 Figure S C NMR spectrum of kadcoccinone B (2) in C 5 D 5 N. 24

25 Figure S22. HSQC spectrum of kadcoccinone B (2) in C 5 D 5 N. 25

26 Figure S23. HMBC spectrum of kadcoccinone B (2) in C 5 D 5 N. 26

27 Figure S24. 1 H 1 H COSY spectrum of kadcoccinone B (2) in C 5 D 5 N. 27

28 Figure S25. ROESY spectrum of kadcoccinone B (2) in C 5 D 5 N. 28

29 Figure S26. 1 H NMR spectrum of kadcoccinone C (3) in C 5 D 5 N. 29

30 Figure S C NMR spectrum of kadcoccinone C (3) in C 5 D 5 N. 30

31 Figure S28. HSQC spectrum of kadcoccinone C (3) in C 5 D 5 N. 31

32 Figure S29. HMBC spectrum of kadcoccinone C (3) in C 5 D 5 N. 32

33 Figure S30. 1 H 1 H COSY spectrum of kadcoccinone C (3) in C 5 D 5 N. 33

34 Figure S31. HSQC-TCOSY spectrum of kadcoccinone C (3) in C 5 D 5 N. 34

35 Figure S32. ROESY spectrum of kadcoccinone C (3) in C 5 D 5 N. 35

36 Figure S33. 1 H NMR spectrum of kadcoccinone C (3) in DMSO. 36

37 Figure S C NMR spectrum of kadcoccinone C (3) in DMSO. 37

38 Figure S35. HSQC spectrum of kadcoccinone C (3) in DMSO. 38

39 Figure S36. HMBC spectrum of kadcoccinone C (3) in DMSO. 39

40 Figure S37. 1 H 1 H COSY spectrum of kadcoccinone C (3) in DMSO. 40

41 Figure S38. ROESY spectrum of kadcoccinone C (3) in DMSO. 41

42 Figure S39. 1 H NMR spectrum of kadcoccinone D (4) in C 5 D 5 N. 42

43 Figure S C NMR spectrum of kadcoccinone D (4) in C 5 D 5 N. 43

44 Figure S41. HSQC spectrum of kadcoccinone D (4) in C 5 D 5 N. 44

45 Figure S42. HMBC spectrum of kadcoccinone D (4) in C 5 D 5 N. 45

46 Figure S43. 1 H 1 H COSY spectrum of kadcoccinone D (4) in C 5 D 5 N. 46

47 Figure S44. ROESY spectrum of kadcoccinone D (4) in C 5 D 5 N. 47

48 Figure S45. 1 H NMR spectrum of kadcoccinone E (5) in C 5 D 5 N. 48

49 Figure S C NMR spectrum of kadcoccinone E (5) in C 5 D 5 N. 49

50 Figure S47. HSQC spectrum of kadcoccinone E (5) in C 5 D 5 N. 50

51 Figure S48. HMBC spectrum of kadcoccinone E (5) in C 5 D 5 N. 51

52 Figure S49. 1 H 1 H COSY spectrum of kadcoccinone E (5) in C 5 D 5 N. 52

53 Figure S50. ROESY spectrum of kadcoccinone E (5) in C 5 D 5 N. 53

54 Figure S51. 1 H NMR spectrum of kadcoccinone F (6) in C 5 D 5 N. 54

55 Figure S C NMR spectrum of kadcoccinone F (6) in C 5 D 5 N. 55

56 Figure S53. HSQC spectrum of kadcoccinone F (6) in C 5 D 5 N. 56

57 Figure S54. HMBC spectrum of kadcoccinone F (6) in C 5 D 5 N. 57

58 Figure S55. 1 H 1 H COSY spectrum of kadcoccinone F (6) in C 5 D 5 N. 58

59 Figure S56. ROESY spectrum of kadcoccinone F (6) in C 5 D 5 N. 59

60 Figure S57. HRESIMS spectrum of kadcoccinone A (1) 60

61 Figure S58. IR spectrum of kadcoccinone A (1) 61

62 Figure S59. ECD (top) and UV (bottom) spectra of kadcoccinone A (1) 62

63 Figure S60. HRESIMS spectrum of kadcoccinone B (2) 63

64 Figure S61. IR spectrum of kadcoccinone B (2) 64

65 Figure S62. UV spectrum of kadcoccinone B (2) Figure S63. ESI spectrum of kadcoccinone C (3) 65

66 Figure S64. HRESIMS spectrum of kadcoccinone C (3) 66

67 Figure S65. IR spectrum of kadcoccinone C (3) 67

68 Figure S66. ECD (top) and UV (bottom) spectra of kadcoccinone C (3) 68

69 Figure S67. HRESIMS spectrum of kadcoccinone D (4) 69

70 Figure S68. IR spectrum of kadcoccinone D (4) 70

71 Figure S69. ECD (top) and UV (bottom) spectra of kadcoccinone D (4) 71

72 Figure S70. HRESIMS spectrum of kadcoccinone E (5) 72

73 Figure S71. IR spectrum of kadcoccinone E (5) 73

74 Figure S72. ECD (top) and UV (bottom) spectra of kadcoccinone E (5) 74

75 Figure S73. HRESIMS spectrum of kadcoccinone F (6) 75

76 Figure S74. IR spectrum of kadcoccinone F (6) 76

77 Figure S75. UV spectrum of kadcoccinone F (6) 77

78 3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k Figure S76. Optimized geometries of conformers of 3 at the B3LYP/6-31G(d) level in the gas phase. 78

79 Table S4. Important thermodynamic parameters (a.u.) of the optimized 3 at B3LYP/6-31G(d) level in the gas phase. species E E =E+ZPE H G 3a b c d e f g h i j k E, E, H, G: total energy, total energy with zero point energy (ZPE), enthalpy, and Gibbs free energy. Table S5. Conformational analysis of 1 at B3LYP/6-31G(d) level. species ΔE a P E % b ΔE c P E % d ΔG e P G % f 3a b c d e f g h i j k a Relative energy, c relative energy with ZPE, and e relative Gibbs free energy in kcal/mol. bdf Conformational distribution calculated by using the respective parameters above at B3LYP/6-31G(d) level in the gas phase. (T=298 K) 79

80 Table S6. Key transitions, oscillator strengths, and rotatory strengths in the ECD spectrum of conformers 3k at B3LYP-SCRF/6-31+G(d,p)//B3LYP/6-31G(d) level in MeOH with PCM. species exited state ΔE (ev) a λ (nm) b f c d R vel 3k R len e 144-> > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > a Excitation energy. b Wavelength. c Oscillator strength. d Rotatory strength in velocity form (10-40 cgs.). e Rotatory strength in length form (10-40 cgs.). 80

81 Table S7. Optimized Z-matrixes of 3 in the gas phase (Å) at B3LYP/6-31G(d) level. 3a 3b C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C H H C C C C C C O O C C H H O O H H C C C C C C C C C C C C C C O O O O C C C C C C O O O O H H H H

82 H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H c 3d C C C C C C

83 C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C H H C C C C C C O O C C H H O O H H C C C C C C C C C C C C C C O O O O C C C C C C O O O O H H H H H H H H H H H H H H H H H H

84 H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H e 3f C C C C C C C C C C C C C C C C C C C C

85 C C C C C C C C C C C C C C C C H H C C C C C C O O C C H H O O H H C C C C C C C C C C C C C C O O O O C C C C C C O O O O H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H

86 H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H g 3h C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C

87 C C H H C C C C C C O O C C H H O O H H C C C C C C C C C C C C C C O O O O C C C C C C O O O O H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H

88 H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H i 3j C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C H H C C C C C C O O C C

89 H H O O H H C C C C C C C C C C C C C C O O O O C C C C C C O O O O H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H