Supporting Information

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1 Supporting Information A Systemic Study on the Biogenic Pathways of Yezo otogirins: Total Synthesis and Antitumor Activities of (±)-Yezo otogirin C and its Structural Analogues Wei Yang, Jingming Cao, Mengxun Zhang, Rongfeng Lan, Lizhi Zhu, Guangyan Du, Shuzhong He, * and Chi-Sing Lee, * Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen University Town, Xili, Shenzhen , China; lizc@pkusz.edu.cn Institute of Research and Continuing Education, Hong Kong Baptist University (Shenzhen), Shenzhen , China School of Pharmaceutical Sciences, Guizhou University, Guiyang, Guizhou , China; she48@wisc.edu Table of Contents X-ray structure of compound 8 (Figure S1)...S2 X-ray structure of compound 9 (Figure S2)...S3 MTT assays of compound 3, 7-9 and 15on human cancer cells (Figure S3 and S4)...S4-5 Flow cytometry cell cycle analysis of Hela cells with compound 3, 7-9 and 15 (Figure S5 and Table S1)...S6-S7 NMR spectra of compound 3, 5-6, 7-9 and S8-S32 NMR spectra of compound 6 and the C6-epmier (a roughly 1:1 mixture)...s33-34 S1

2 X-ray structure of compound 8 Figure S1. X-ray structure of compound 8. The crystals of compound 8 were obtained by recrystallization from n-hexane S2

3 X-ray structure of compound 9 Figure S2. X-ray structure of compound 9. The crystals of compound 9 were obtained by recrystallization from n- hexane S3

4 MTT assays of compound 3, 7-9 and 15 on human cancer cells (a) (b) (c) Figure S3. Cell growth inhibitory effects of compound 3, 7-9 and 15 on (a) HeLa, (b) SMMC-7721 and (c) MGc80-3. S4

5 Cell cycle analysis of Hela cells with compound 3, 7-9 and 15 Figure S4. Cell cycle analysis of HeLa cells treated with compound 3, 7-9 and 15. The representative microscopic images of HeLa cells incubated with the chemicals, DMSO panel were parallel performed as control. Scale bar = 80 μm. S5

6 Flow cytometry analysis the cell cycle of HeLa cells treated with compound 3, 7-9 and 15 Figure S5. The representative histograms results of the Flow cytometry analysis (graphed by FlowJo 7.6.1). 2N and 4N indicate the diploid cells and replicated diploid cells, respectively. S6

7 Table S1. Flow cytometry data of the cell cycle analysis for Figure S5 (a-e) a G1 G2 S sub-g1 DMSO 64.8± ± ± ± (10 μm) 65.82± ± ± ± (20 μm) 71.61± ± ± ± (30 μm) 60.98± ± ± ± (40 μm) 63.37± ±0.98 ** 25.95± ± (50 μm) 62.02± ±0.93 ** 27.45± ± (60 μm) 56.99± ±0.63 ** 33.38± ± (10 μm) 67.77± ± ± ± (20 μm) 64.56± ± ± ± (30 μm) 64.49± ± ± ± (40 μm) 68.85± ±0.93 ** 19.83± ± (50 μm) 64.57± ±0.99 ** 22.97± ± (60 μm) 67.69± ±0.64 ** 21.46± ± (10 μm) 72.74±0.52 ** 8.78± ± ± (20 μm) 71.59±0.96 ** 5.74± ± ± (30 μm) 77.40±1.34 ** 7.47± ± ± (40 μm) 79.74±0.41 ** 6.62± ± ± (50 μm) 83.21±2.71 ** 7.04± ± ±0.35 8(60 μm) 80.65±1.23 ** 11.22±0.36 ** 8.14± ± (10 μm) 64.51±2.10 * 6.84± ± ±2.89 9(20 μm) 63.61±0.30 ** 9.08± ± ± (30 μm) 65.43±2.54 ** 0.68± ± ± (40 μm) 68.42±1.09 ** 1.45± ± ± (50 μm) 68.27±2.14 ** 0.47± ± ± (60 μm) 68.87±2.81 ** 0.49± ± ± (10 μm) 62.92± ±0.60 * 27.54± ± (20 μm) 67.14± ±0.80 * 24.21± ± (30 μm) 73.33± ±2.28 ** 12.47± ± (40 μm) 62.27± ±1.71 ** 9.33± ± (50 μm) 47.47± ±0.51 ** 8.73± ± (60 μm) 29.22± ±4.00 ** 18.41± ±4.93 a All values are the average of three assays (average ± S.D., * P<0.05, ** P<0.01). Tri-duplicates were performed for each concentration of the compounds. The sub-g1 values were showed independently, that is the percent from the whole cell population in the FACS sorting (sub G1%=sub-G1/(sub-G1+G1+G2+S) 100%). S7

8 1 H and 13 C NMR of compound 12 S8

9 1 H and 13 C NMR of compound 13 S9

10 1 H and 13 C NMR spectra of compound 5 (a mixture of β-keto esters) S10

11 1 H and 13 C NMR of compound 14 (single diastereomer) S11

12 1 H and 13 C NMR spectra of compound 6 S12

13 HSQC and COSY spectra of compound 6 S13

14 NOESY spectrum and the stereochemistry assignment of compound 6 S14

15 1 H and 13 C NMR spectra of compound 7 S15

16 HSQC and COSY spectra of compound 7 S16

17 NOESY spectra and the stereochemistry assignment of compound 7 S17

18 1 H and 13 C NMR spectra of compound 8 S18

19 HSQC and COSY spectra of compound 8 S19

20 NOESY spectra and the stereochemistry assignment of compound 8 S20

21 1 H and 13 C NMR spectra of compound 15 S21

22 HSQC and COSY spectra of compound 15 S22

23 NOESY spectra and the stereochemistry assignment of compound 15 S23

24 1 H and 13 C NMR spectra of compound 16 S24

25 1 H and 13 C NMR spectra of compound 17 S25

26 1 H and 13 C NMR spectra of compound 18 (a mixture of alcohol diastereomers) S26

27 1 H and 13 C NMR spectra of compound 9 S27

28 HSQC and COSY spectra of compound 9 S28

29 NOESY spectra and the stereochemistry assignment of compound 9 S29

30 1 H and 13 C NMR spectra of (±)-yezo otogirin C (3) S30

31 HSQC and COSY spectra of (±)-yezo otogirin C (3) S31

32 NOESY spectra and the stereochemistry assignment of (±)-yezo otogirin C (3) S32

33 1 H NMR and 13 C NMR spectra of 6 and epi-6-6 (a roughly 1:1 mixture) S33

34 NOESY spectra of the 1:1 mixture and the partial stereochemistry assignment of epi-6-6 S34