Synthesis of Glaucogenin D, a Structurally Unique. Disecopregnane Steroid with Potential Antiviral Activity

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1 Supporting Information for Synthesis of Glaucogenin D, a Structurally Unique Disecopregnane Steroid with Potential Antiviral Activity Jinghan Gui,* Hailong Tian, and Weisheng Tian* Key Laboratory of Synthetic Chemistry of Natural Substances, Shanghai Insitute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, China.

2 Table of Contents 1. General Experimental Procedure... S2 2. Full Synthetic Sequence to Glaucogenin D from hirundigenin-type intermediate 6... S3 3. Experimental Section... S3 4. Comparison of 1 H- and 13 CNMR Data for Natural vs Synthetic Glaucogenin D... S15 5. NMR Spectra... S16 S1

3 1. General Experimental Procedure: All reactions utilizing air- or moisture-sensitive reagents were carried out in flame-dried glassware under an argon atmosphere, unless otherwise stated. CH 2 Cl 2, DMF, t BuOH and THF were distilled prior to use according to the standard protocols. Other reagents were purchased and used as received without further purification unless otherwise stated. Reactions were magnetically stirred and monitored by thin layer chromatography (TLC) with mm pre-coated silica gel (10-40 μm) plates. Compounds were visualized with UV light and/or by staining with ethanolic phosphomolybdic acid (PMA) followed by heating on a hot plate. Flash chromatography was performed with silica gel ( mesh) under pressure. Yields refer to chromatographically and spectroscopically ( 1 H NMR) homogeneous compounds, unless otherwise stated. NMR spectra were recorded on Varian-300, Bruker-400 and Bruker-500 spectrometers in CDCl 3 with TMS as the internal standard, unless otherwise stated. Chemical shifts (δ) are given in ppm relative to residual chloroform (δ 7.26 for 1 H NMR and 77.2 for 13 C NMR), coupling constants (J) in Hz. Multiplicity is indicated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. IR spectra were collected on Avatar 330 FT-IR spectrometer. Melting points were determined on SGW X-4 microscopic melting point apparatus and were uncorrected. Optical rotations were determined on JASCO P-1030 Polarimeter in the solvent indicated. High-resolution mass spectra were recorded on IonSpec 4.7 Tesla FTMS or Bruker Daltonics, Inc. APEXIII 7.0 TESLA FTMS. S2

4 3. Full Synthetic Sequence to Glaucogenin D from Hirundigenin-type Intermediate 6: 4. Experimental Section: A solution of 7 (15 mg, mmol) and tetraphenylporphyrin (TPP) (ca. 1 mg) in dry DCM (5 ml) was irradiated with an incandescent lamp (200 W) while oxygen was being passed through the solution. The mixture was stirred at 0 o C for 1 h. Evaporation of the solvent under reduced pressure and flash chromatography of the residue (EtOAc: PE=1:3) afforded 9 (16 mg, >99%) as a white solid. [α] 26 D 10.9 (c 0.65, CHCl 3 ); mp o C; IR (KBr, cm -1 ) 3419, 2931, 2857, 1748, 1428, 1384, 1104, 702, 508; 1 H NMR (300 MHz, CDCl 3 ): 0.73 (s, 3H), 1.04 (s, 9H), 1.61 (s, 3H), 2.06 (s, 3H), 2.89 (d, J = 6.6 Hz, 1H), 3.49 (d, J = 9.3 Hz, 1H), 3.56 (m, 1H), 3.77 (d, J = 9.3 Hz, 1H), 4.65 (d, J = 6.9 Hz, 1H), 5.89 (m, 1H), 6.33 (s, 3H), S3

5 (m, 6H), 7.61 (s, 1H), 7.66 (br d, J = 6.9 Hz, 4H); 13 C NMR (100 MHz, CDCl 3 ) δ 169.7, 136.0, 135.0, 134.9, 131.2, 129.7, 127.7, 124.7, 120.0, 113.3, 99.1, 87.4, 72.6, 72.4, 60.4, 59.0, 45.8, 40.0, 38.1, 37.2, 34.6, 31.7, 29.2, 27.3, 25.9, 25.6, 21.5, 21.4, 19.4, 13.2; HRMS (ESI) m/z (M+Na) + calcd for C 39 H 50 O 8 SiNa , obsd A solution of 7 (128 mg, 0.2 mmol) and tetraphenylporphyrin (TPP) (ca. 1 mg) in dry DCM (20 ml) was irradiated with an incandescent lamp (200 W) while oxygen was being passed through the solution. After the mixture was stirred at 0 o C for 1 h, the solvent was removed in vacuo. To another solution of FeSO 4 7H 2 O (115 mg, 0.4 mmol) and I 2 (106 mg, 0.4 mmol) in 2 ml MeOH was added under argon a solution of above mixtures in MeOH (6 ml). After stirred at RT for 1 h, the reaction mixture was quenched with saturated Na 2 S 2 O 3 solution (15 ml) and extracted with DCM (3 20 ml). The combined organic layers were washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. To the crude residue was added DCM (2 ml), DBU (0.3 ml, 2 mmol) and the mixture was stirred at 60 o C for 2 h. After the reaction mixture was cooled to RT, 5% HCl (10 ml) was added and the water phase was extracted with DCM (3 20 ml). The combined organic layers were washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. The crude residue was purified by flash chromatography (EtOAc: PE=1:10) to afford 11 (109 mg, 83% over 3 steps) as a white solid. [α] 25 D 31.8 (c 1.60, CHCl 3 ); mp o C; IR (KBr, cm -1 ) 2931, 2857, 1764, 1729, 1427, 1110, 702, 508; 1 H NMR (400 MHz, CDCl 3 ): 0.73 (s, 3H), 1.08 (s, 9H), 1.66 (s, 3H), 2.15(s, 3H), 2.58 (m, 1H), 3.58 (d, J = 8.0 Hz, 1H), 3.60 (m, 1H), 5.48 (dd, J = 8.0, 4.4 Hz, 1H), 6.00 (m, 1H), 6.19 (s, 1H), 6.32 (d, J = 4.4 Hz, 1H), (m, 6H), 7.70 (m, 4H); 13 C NMR (101 MHz, CDCl 3 ) δ 170.8, 169.6, 142.1, 135.8, 134.7, 134.0, 130.1, 129.5, 127.5, 117.3, 115.2, 97.9, 79.8, 72.0, 54. 8, 51.3, S4

6 39.3, 37. 9, 36.1, 34.8, 31.8, 29.6, 27.5, 27.0, 25.0, 22.9, 21.1, 19.1, 11.4; HRMS (ESI) m/z (M+Na) + calcd for C 39 H 48 O 7 SiNa , obsd A solution of 7 (99 mg, 0.15 mmol) and tetraphenylporphyrin (TPP) (ca. 1 mg) in dry DCM (10 ml) was irradiated with an incandescent lamp (200 W) and oxygen was passed through the solution. After the mixture was stirred at 0 o C for 1 h, the solvent was removed in vacuo. To another solution of FeSO 4 7H 2 O (83 mg, 0.3 mmol) and TEMPO (47 mg, 0.3 mmol) in 1 ml MeOH was added under argon a solution of above mixture in MeOH (3 ml). After stirred at RT for 1 h, the reaction mixture was quenched with 5% HCl solution (10 ml) and extracted with DCM (3 20 ml). The combined organic layers were washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. The crude residue was purified by flash chromatography (EtOAc: PE=1:10) to afford 13 (87 mg, 71% over 2 steps) as a white solid. [α] 23 D 10.3 (c 1.65, CHCl 3 ); mp o C; IR (KBr, cm -1 ) 2932, 2856, 1759, 1728, 1375, 1108, 702, 507; 1 H NMR (400 MHz, CDCl 3 ): 0.70 (s, 3H), 0.94 (s, 3H), 0.98 (s, 3H), 1.05 (s, 9H), 1.09 (s, 6H), 1.68 (s, 3H), 2.09 (s, 3H), 2.98 (d, J = 8.8 Hz, 1H), 3.16 (m, 1H), 3.57 (m, 1H), 4.08 (d, J = 6.0 Hz, 1H), 4.12 (d, J = 6.0 Hz, 1H), 5.67 (d, J = 7.2 Hz, 1H), 6.22 (m, 1H), 6.26 (s, 1H), (m, 6H), 7.67 (m, 4H); 13 C NMR (101 MHz, CDCl 3 ) δ 170.4, 169.2, 135.7, 134.7, 134.6, 134.5, 133.3, 129.5, 127.5, 119.5, 100.9, 92.9, 77.7, 75.5, 72.0, 60.3, 59.5, 59.3, 49.7, 40.6, 40.5, 39.4, 37.8, 36.0, 34.8, 34.2, 33.8, 31.8, 29.9, 27.0, 26.7, 26.2, 21.1, 20.9, 20.8, 19.1, 16.9, 11.4; HRMS (ESI) m/z (M+Na) + calcd for C 48 H 67 NO 8 SiNa , obsd S5

7 To a solution of 12 (15 mg, 0.02 mmol) and dibenzo-18-crown-6 (18 mg, 0.05 mmol) in dry DCM (1 ml) was added KMnO 4 (6.3 mg, 0.04 mmol) at 0 o C. After stirred at this temperature for 2 h, the reaction mixture was warmed to RT and stirring was continued for another 3.5 h. Then the reaction mixture was quenched successively with saturated NaHSO 3 solution, 5% HCl solution and extracted with DCM (3 10 ml). The combined organic layers were washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. The crude residue was purified by flash chromatography (EtOAc: PE=1:10 1:2) to afford 15 (11 mg, 70%, 75% brsm) as a white solid, together with unreacted starting material (1 mg, 7%). [α] 26 D 37.5 (c 0.85, CHCl 3 ); mp o C; IR (KBr, cm -1 ) 3493, 2932, 2857, 1732, 1214, 1109, 702, 506; 1 H NMR (400 MHz, CDCl 3 ): 0.79 (s, 3H), 0.97 (s, 1H), 0.98 (s, 1H), 1.05 (s, 9H), 1.09 (s, 3H), 1.10 (s, 3H), 1.65 (s, 3H), 2.83 (m, 1H), 2.93 (d, J = 9.6 Hz, 1H), 3.59 (m, 1H), 3.85 (s, 1H), 3.93 (t, J = 8.8 Hz, 1H), 4.10 (m, 2H), 4.19 (dd, J = 9.6, 8.0 Hz, 1H), 4.24 (d, J = 10.0 Hz, 1H), 5.65 (q, J = 8.8 Hz, 1H), (m, 6H), 7.66 (m, 4H); 13 C NMR (101 MHz, CDCl 3 ) δ 175.9, 135.7, 134.8, 134.6, 129.5, 127.5, 116.9, 93.2, 80.0, 77.2, 73.9, 71.9, 69.2, 67.9, 59.7, 59.4, 58.5, 57.9, 40.8, 40.7, 39.4, 38.1, 36.6, 35.1, 34.9, 34.3, 34.1, 31.1, 27.0, 24.5, 24.2, 21.7, 21.3, 21.1, 19.1, 16.9, 14.4; HRMS (ESI) m/z (M+H) + calcd for C 46 H 68 NO 8 Si , obsd To a solution of 15 (18 mg, mmol) in dry DMF (1 ml) was added PDC (87 mg, 0.23 mmol) at RT. After stirred at RT for 28 h, the reaction mixture was quenched with water and extracted with DCM (3 10 ml). The combined organic layers were washed with water (3 10 ml), brine, dried over Na 2 SO 4, and concentrated in vacuo. The crude residue was purified by flash chromatography (EtOAc: PE=1:5) to afford 16 (15 mg, 83%) as a white solid. [α] 22 D 36.7 (c 1.75, CHCl 3 ); mp o C; IR (KBr, cm -1 ) 3479, 2932, 2856, 1732, 1249, 1111, 702, 501; 1 H NMR (400 MHz, CDCl 3 ): 0.71 (s, 3H), 0.96 (s, 1H), 0.98 (s, 1H), 1.06 (s, 9H), 1.08 (s, 3H), 1.10 (s, S6

8 3H), 1.64 (s, 3H), 2.26 (m, 1H), 2.64 (dd, J = 18.4, 7.2 Hz, 1H), 2.81 (m, 1H), 2.92 (d, J = 10.0 Hz, 1H), 3.63 (m, 1H), 3.92 (t, J = 9.2 Hz, 1H), 4.05 (d, J = 10.0 Hz, 1H), 4.08 (s, 1H), 4.23(m, 2H), 5.74 (q, J = 8.8 Hz, 1H), (m, 6H), 7.66 (m, 4H); 13 C NMR (101 MHz, CDCl 3 ) δ 204.6, 172.7, 135.7, 134.6, 134.4, 129.6, 127.6, 116.9, 93.2, 80.2, 77.2, 74.1, 71.6, 69.1, 60.9, 59.8, 59.5, 57.5, 40.8, 40.7, 40.1, 37.8, 36.3, 35.3, 34.4, 34.1, 31.3, 27.0, 24.5, 23.8, 21.4, 21.2, 21.0, 19.1, 16.9, 13.3; HRMS (ESI) m/z (M+Na) + calcd for C 46 H 65 NO 8 SiH , obsd To a solution of i Pr 2 NH (0.26 ml, 1.83 mmol) in 1 ml anhydrous THF was added at 0 o C a solution of 2.5 M n-buli (in hexane) (0.78 ml, 1.83 mmol). After stirred at this temperature for 30 min, the mixture was cooled to -78 o C and a solution of 16 (70 mg, mmol) in 1.5 ml THF and 0.2 ml HMPA was added. After 1 h, TMSCl (0.23 ml,1.80 mmol)was added and stirring was continued for 30 min. Then the mixture was warmed to 0 o C and stirred for another 1 h before being quenched with saturated NaHCO 3 solution and extracted with EtOAc (3 20 ml). The combined organic layers were washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. The crude residue of silyl enol ether 17 was used in the next step without further purification. 1 H NMR (400 MHz, CDCl 3 ): 0.05 (s, 9H), 0.14 (s, 9H), 0.70 (s, 3H), 1.01 (s, 3H), 1.07 (s, 9H), 1.09 (s, 3H), 1.63 (s, 3H), (m, 1H), 3.08 (d, J = 10.0 Hz, 1H), (m, 1H), 3.89 (t, J = 9.6 Hz, 1H), 4.06 (d, J = 10.0 Hz, 1H), 4.12 (t, J = 8.0 Hz, 1H), 4.22 (d, J = 10.0 Hz, 1H), 4.51 (d, J = 2.8 Hz, 1H), 5.36 (dt, J = 8.0, 10.0 Hz, 1H), (m, 6H), 7.68 (m, 4H); HRMS (ESI) m/z (M+H) + calcd for C 52 H 82 NO 8 Si , obsd S7

9 Condition A: To the crude mixture of 17 (prepared from 16 (70 mg, mmol) as described above) in 2 ml anhydrous THF was added NBS (95 mg, mmol) at RT. After stirred at this temperature for 3 h, the reaction mixture was quenched with saturated NaHCO 3 solution, and extracted with DCM (3 30 ml). The combined organic layers were washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. The crude residue was purified by flash chromatography (EtOAc: PE=1:15 1:10) to afford 18 (90 mg, >99%) as a white solid. Condition B: To the crude mixture of 17 (prepared from 16 (7 mg, mmol) as described above) in 1 ml anhydrous THF was added PhSeBr (21 mg, mmol) at RT. After stirred at this temperature for 11 h, the reaction mixture was quenched with saturated NaHCO 3 solution, and extracted with DCM (3 10 ml). The combined organic layers were washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. The crude residue was purified by flash chromatography (EtOAc: PE=1:15 1:10) to afford 18 (10 mg, >99%) as a white solid. Compound 18: [α] 28 D 23.7 (c 1.40, CHCl 3 ); mp o C; IR (KBr, cm -1 ) 2930, 2856, 1744, 1730, 1471, 1252, 1111, 702, 503; 1 H NMR (300 MHz, CDCl 3 ): 0.15 (s, 9H), 0.68 (s, 3H), 1.00 (s, 6H), 1.08 (s, 9H), 1.09 (s, 3H), 1.10 (s, 3H), 1.64 (s, 3H), 2.19 (m, 1H), (m, 1H), (m, 1H), 2.92 (d, J = 10.0 Hz, 1H), (m, 1H), 3.72 (d, J = 11.6 Hz, 1H), 3.86 (t, J = 9.2 Hz, 1H), 4.01 (d, J = 10.4 Hz, 1H), (m, 2H), 5.68 (q, J = 9.2 Hz, 1H), (m, 6H), 7.68 (m, 4H); 13 C NMR (101 MHz, CDCl 3 ) δ , , , , , , , , , , 83.94, 73.48, 71.78, 69.28, 62.56, 60.20, 59.88, 57.46, 47.73, 44.07, 41.02, 40.91, 39.10, 36.75, 35.48, 34.98, 34.28, 31.37, 29.87, 27.17, 24.74, 22.13, 21.36, 21.23, 19.27, 17.05, 13.99, 2.73; HRMS (ESI) m/z (M+Na) + calcd for C 49 H 72 BrNO 8 Si 2 Na , obsd S8

10 To the crude mixture of 17 (prepared from 16 (157 mg, 0.2 mmol) as described above) in 5 ml anhydrous DCM was added PhSeCl (384 mg, 2 mmol) at RT. After stirred at this temperature for 2.5 h, the reaction mixture was quenched with saturated NaHCO 3 solution, and extracted with DCM (3 30 ml). The combined organic layers were washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. The crude residue was purified by flash chromatography (EtOAc: PE=1:8) to afford 20 (201 mg, 99%) as a white solid. [α] 27 D 26.4 (c 2.50, CHCl 3 ); mp o C; IR (KBr, cm -1 ) 2932, 2857, 1747,1723, 1250, 1111, 847, 702, 509; 1 H NMR (400 MHz, CDCl 3 ): 0.21 (s, 9H), 0.64 (s, 3H), 0.98 (s, 3H), 0.99 (s, 3H), 1.07 (s, 9H), 1.09 (s, 3H), 1.10 (s, 3H), 1.64 (s, 3H), (m, 1H), 2.48 (m, 1H), (m, 3H), (m, 1H), 3.87 (t, J = 9.2 Hz, 1H), 4.03 (d, J = 10.0 Hz, 1H), (m, 2H), 5.73 (q, J = 9.2 Hz, 1H), (m, 3H), (m, 6H), (m, 2H), (m, 4H); 13 C NMR (101 MHz, CDCl 3 ) δ , , , , , , , , , , , , , , 93.45, 84.04, 77.29, 73.21, 72.28, 69.29, 62.77, 59.97, 59.62, 57.47, 51.25, 43.61, 41.11, 40.97, 38.95, 36.64, 35.55, 35.07, 34.40, 31.45, 29.87, 27.18, 24.76, 23.60, 22.16, 21.43, 21.25, 19.29, 17.09, 13.54, 3.20; LR-MS (ESI) m/z (M+Na) + : (Acquisition of HRMS data (ESI or MALDI) was met with failure possiblely due to the high MW of the molecule). To a solution of 20 (61 mg, 0.06 mmol) in 3 ml CH 3 CN was added an aqueous 40% HF solution (0.03 ml, 0.6 mmol) and the resulting solution was stirred at RT for 1 day. S9

11 Then the reaction mixture was quenched with saturated NaHCO 3 solution (15 ml) and extracted with EtOAc (3 20 ml). The combined organic layers were washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. The crude residue of 21 was dissolved in DCM (10 ml) and O 3 was passed through the solution while cooling at 78 o C. After 1 min, a blue color appeared and the ozone bubbler was replaced with an argon bubbler to give a colorless solution. Then 0.01 ml i Pr 2 NH was added and the solution was warmed to RT. To the crude mixture, obtained by evaporation of the solvent under reduced pressure, in 2 ml toluene was added 0.01 ml i Pr 2 NH and the resulting solution was stirred at 60 o C for 1 h. Evaporation of the solvent under reduced pressure and flash chromatography of the residue (EtOAc: PE=1:1 2:1) afforded 22 (17 mg, 52%) as a white solid. Compound 21: [α] 27 D 61.7 (c 1.00, CHCl 3 ); mp o C; IR (KBr, cm -1 ) 3536, 3311, 2934, 2870, 1745, 1706, 1270, 738; 1 H NMR (400 MHz, CDCl 3 ): 0.72 (s, 3H), 1.01 (s, 3H), 1.02 (s, 3H), 1.10 (s, 3H), 1.12 (s,3h), 1.65 (s, 3H), 2.40 (m, 1H), 2.63 (m, 1H), 2.92 (m, 2H), 3.00 (d, J = 11.2 Hz, 1H), 3.69 (m, 1H), 3.94 (t, J = 9.2 Hz, 1H), 4.07 (m, 2H), 4.24 (m, 2H), 5.82 (q, J = 8.4 Hz, 1H), 7.28 (m, 3H), 7.65 (m, 2H); 13 C NMR (101 MHz, CDCl 3 ) δ , , , , , , , 93.46, 81.11, 74.15, 70.30, 69.46, 61.26, 60.12, 59.82, 57.83, 50.70, 44.15, 40.98, 40.88, 38.31, 36.40, 35.54, 34.52, 34.25, 31.18, 24.59, 24.29, 21.67, 21.39, 21.22, 17.07, 13.38; HRMS (ESI) m/z (M+Na) + calcd for C 36 H 51 NO 8 SeNa , obsd Compound 22: [α] 26 D 71.4 (c 0.85, CHCl 3 ); mp o C; IR (KBr, cm -1 ) 3467, 2933, 1739, 1674, 1466, 1239, 1061, 877; 1 H NMR (400 MHz, CDCl 3 ): 0.99 (s, 3H), 1.00 (s, 3H), 1.11 (s, 6H), 1.31 (s, 3H), 1.61 (s, 3H), 2.55 (m, 2H), 2.64 (m, 1H), 2.85 (d, J = 9.2 Hz, 1H), 3.35 (m, 1H), 3.73 (m, 1H), 3.95 (s, 1H), 4.00 (m, 2H), 4.15 (m, 2H), 5.89 (s, 1H), 5.90 (m, 1H); 13 C NMR (101 MHz, CDCl 3 ) δ , , , , , 79.73, 74.77, 73.18, 70.48, 60.24, 59.82, 59.59, 53.44, 43.14, 40.97, 40.83, 40.22, 38.25, 34.27, 34.13, 31.81, 27.00, 24.23, 24.10, 21.53, 21.19, 20.75, 17.09; HRMS (ESI) m/z (M+Na) + calcd for C 30 H 45 NO 8 Na , obsd S10

12 Preparation of then SmI 2 solution in THF: To a suspension of powdered Sm (105 mg, mmol) in 2 ml THF was added CH 2 I 2 (27 μl, mmol). The mixture was stirred at room temperature for 1 h, observing the appearance of a dark blue color. To a solution of 22 (16 mg,0.029 mmol) in THF (1 ml) and t-buoh (0.1 ml) was added at -78 o C a solution of SmI 2 in THF (1 ml, ca mmol) freshly prepared as described above. The reaction mixture was stirred at this temperature for 10 min and opened to air untill a yellow color appeared. Then a saturated NaHCO 3 solution (15 ml) was added, and the mixture was warmed to RT and extracted with DCM (3 15 ml). The combined organic layers were washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. To this crude mixture in 2 ml MeOH at 0 o C was added sequentially CeCl 3 7H 2 O (32 mg, mmol), NaBH 4 (4 mg, mmol). After stirred at 0 o C for 1 h, the reaction mixture was quenched with saturated NH 4 Cl solution (15 ml) and extracted with DCM (3 15 ml). The combined organic layers were washed with brine, dried over Na 2 SO 4, and concentrated in vacuo. The crude residue was purified by flash chromatography (EtOAc: PE=1:1 2:1) to afford 23 (9 mg, 58% for 2 steps) as a white solid. [α] 27 D -3.5 (c 0.65, CHCl 3 ); mp o C; IR (KBr, cm -1 ) 3432, 2930, 2854, 1736, 1464, 1163, 1048, 828; 1 H NMR (400 MHz, CDCl 3 ): 1.01 (s, 3H), 1.13 (s, 3H), 1.14 (s, 3H), 1.26 (s, 6H), 1.65 (s, 3H), (m, 1H), (m, 2H), 2.82 (d, J = 9.2 Hz, 1H), (m, 1H), (m, 1H), 3.99 (dd, J = 10.0, 5.6 Hz, 1H), 4.11 (dd, J = 9.6, 6.8 Hz, 1H), 4.17 (d, J = 9.6 Hz, 1H), 4.24 (d, J = 9.6 Hz, 1H), 4.64 (br d, J = 9.6 Hz, 1H), 5.41 (s, 1H), 5.86 (dt, J = 9.6, 6.4 Hz, 1H); 13 C NMR (101 MHz, CDCl 3 ) δ , , , , 93.20, 77.11, 72.36, 71.57, 70.48, 68.20, 60.07, 59.69, 59.36, 49.21, 48.30, 41.67, 40.96, 40.87, 38.83, 36.23, S11

13 34.24, 34.21, 31.37, 24.88, 24.29, 22.72, 21.63, 21.31, 18.03, 17.13; HRMS (ESI) m/z (M+H) + calcd for C 30 H 48 NO , obsd To a solution of 23 (10 mg, mmol) in 2 ml toluene was added 0.01 ml i Pr 2 NH and the resulting solution was stirred at 120 o C for 1 h. Evaporation of the solvent under reduced pressure and flash chromatography of the residue (EtOAc: PE=1:1 EA) afforded 24 (6 mg, 85%) as a white solid. [α] 28 D 2.6 (c 0.30, CHCl 3 ); mp o C; IR (KBr, cm -1 ) 3416, 2930, 2854, 1725, 1151, 1047, 809; 1 H NMR (400 MHz, CDCl 3 ): 1.09 (s, 3H), 1.55 (s, 3H), 3.22 (d, J = 8.0 Hz, 1H), (m, 1H), 4.10 (dd, J = 6.4, 4.0 Hz, 1H), 4.22 (dd, J = 10.4, 6.4 Hz, 1H), 4.39 (d, J = 12.8 Hz, 1H), (m, 2H), 5.05 (dd, J = 12.4, 1.2 Hz, 1H), 5.39 (s, 1H), (m, 1H); 13 C NMR (101 MHz, CDCl 3 ) δ , , , , , , 78.31, 71.49, 71.21, 71.11, 70.25, 56.60, 50.67, 50.60, 41.73, 38.18, 37.10, 31.63, 29.36, 23.21, 19.94; HRMS (ESI) m/z (M+Na) + calcd for C 21 H 28 O 6 Na , obsd To a solution of 24 (3 mg, mmol) in 1 ml n PrOH was added RhCl(PPh 3 ) 3 1 mg and the resulting solution was stirred at 120 o C for 2 h. Evaporation of the solvent under reduced pressure and flash chromatography of the residue (EtOAc: PE=2:1) afforded glaucogenin D, 4 (2 mg, 66%) as a white solid. [α] 29 D 73.6 (c 0.09, CHCl 3 ); m.p ; IR (KBr, cm -1 ) 3425, 2929, 2855, 1737, 1383, 1083, 807, 609; 1 H NMR (400 MHz, CDCl 3 ): 0.99 (s, 3H), 1.54 (s, 3H), (m, 1H), 3.45 (d, J = 1 Since we can not obtain a crystal form of glaucogenin D, the m.p. data herein is much smaller than the reported data (m.p o C, white needle crystal from acetone-h 2 O). See: Dou, J.; Bi, Z.-M.; Zhang, Y.-Q.; Li, P. Chin. J. Nat. Med. 2006, 4, 192. S12

14 7.6 Hz, 1H), (m, 1H), 3.85 (t, J = 9.2 Hz, 1H), 4.17 (t, J = 8.8 Hz, 1H), 4.62 (br d, J = 10.0 Hz, 1H), (m, 1 H), 5.39 (s, 1H), 6.26 (s, 1H); 1 H NMR (400 MHz, Py): 0.91 (s, 3H), 1.51 (s, 3H), 3.59 (d, J = 8.0 Hz, 1H), 3.84 (m, 1H), 3.91 (dd, J = 9.6, 8.8 Hz, 1H), 4.11 (dd, J = 8.4, 7.2 Hz, 1H), 5.07 (d, J = 9.6 Hz, 1H), 5.43 (dt, J = 9.2, 7.6 Hz, 1H), 5.75 (s, 1H), 6.46 (s, 1H); 13 C NMR (101 MHz, Py) δ , , , , , , 75.84, 71.09, 67.97, 67.87, 56.39, 51.40, 50.81, 42.81, 38.79, 36.58, 32.40, 30.22, 24.80, 23.73, 17.92; HRMS (ESI) m/z (M-H) - calcd for C 21 H 27 O , obsd To a solution of 12 (8 mg, 0.01 mmol) in 1 ml toluene was added 0.01 ml i Pr 2 NH and the resulting solution was stirred at 120 o C for 2 h. Evaporation of the solvent under reduced pressure and flash chromatography of the residue (EtOAc: PE=1:15 1:5) afforded 25 (6 mg, 95%) as a white solid. [α] 24 D 2.7 (c 0.85, CHCl 3 ); mp o C; IR (KBr, cm -1 ) 2930, 2856, 1715, 1427, 1230, 1109, 702, 508; 1 H NMR (400 MHz, CDCl 3 ): 0.84 (s, 3H), 1.05 (s, 9H), 1.51 (s, 3H), 2.33 (m, 1H), 2.51 (m, 1H), 3.16 (d, J = 7.6 Hz, 1H), (m, 1H), 4.10 (dd, J = 10.8, 4.0 Hz, 1H), 4.19 (dd, J = 10.8, 6.0 Hz, 1H), 4.31 (d, J = 12.4 Hz, 1H), 4.42 (d, J = 12.4 Hz, 1H), 5.18 (m, 1H), 5.59 (m, 1H), 5.89 (m, 1H), (m, 6H), 7.66 (m, 4H); 13 C NMR (101 MHz, CDCl 3 ) δ 173.4, 142.0, 136.4, 135.7, 134.7, 134.6, 129.5, 127.9, 127.5, 126.6, 115.6, 79.9, 72.0, 70.8, 70.3, 56.9, 51.3, 40.3, 37.7, 37.2, 34. 8, 31.3, 28.8, 28.7, 27.0, 23.0, 19.1, 14.0; HRMS (ESI) m/z (M+Na) + calcd for C 37 H 46 O 5 SiNa , obsd S13

15 Condition A: To a solution of 25 (14 mg, mmol) in 1 ml anhydrous toluene was added 0.01 ml DIPEA and RuCl 2 (PPh 3 ) 2 (19 mg, 0.02 mmol) and the resulting solution was stirred at 120 o C for 24 h. Evaporation of the solvent under reduced pressure and flash chromatography of the residue (EtOAc: PE=1:10) afforded 10 (10 mg, 71%, >99% brsm) as a white solid, together with unreacted starting material (4 mg, 39%). Condition B: To a solution of 25 (5 mg, mmol) in 1 ml n PrOH was added RhCl(PPh 3 ) 3 (1 mg, mmol) and the resulting solution was stirred at 110 o C for 1.5 h. Evaporation of the solvent under reduced pressure and flash chromatography of the residue (EtOAc: PE=1:10) afforded 10 (5mg, >99%) as a white solid. Compound 10: [α] 26 D 27.9 (c 1.75, CHCl 3 ); mp o C; IR (KBr, cm -1 ) 2931, 2856, 1723, 1654, 1110, 865; 1 H NMR (400 MHz, CDCl 3 ): 0.68 (s, 3H), 1.05 (s, 9H), 1.26 (s, 3H), 2.06 (m, 1H), 2.56 (m, 1H), 3.42 (d, J = 7.6 Hz, 1H), (m, 1H), 3.79 (t, J = 9.2 Hz, 1H), 4.21 (t, J = 7.6 Hz, 1H), 5.36 (dt, J = 9.2, 7.6 Hz, 1H), 6.00 (br s, 1H), 6.25(s, 1H), (m, 6H), 7.67 (br d, J = 7.2 Hz, 4H); 13 C NMR (101 MHz, CDCl 3 ) δ 170.7, 143.2, 135.7, 134.7, 134.6, 130.1, 129.5, 127.5, 117.1, 113.8, 75.1, 72.0, 67.7, 55.4, 51.8, 39.3, 37.9, 36.1, 34.8, 31.8, 29.6, 28.4, 27.0, 24.6, 23.8, 19.1, 11.4; HRMS (MALDI) m/z (M+Na) + calcd for C 37 H 46 O 5 SiNa , obsd S14

16 5. Comparison of 1 H- and 13 CNMR Data for Natural vs Synthetic Glaucogenin D. Comparison of 1 H NMR chemical shifts (Py-d5) Proton Natural Glaucogenin D 2 Synthetic Glaucogenin D (400 MHz) Δδ 18-CH 6.46 (s) 6.46 (s) 0 6-CH 5.75 (d, J = 1.5 Hz) 5.75 (s) 0 16-CH 5.43 (dd, J = 9.0, 7.8 Hz) 5.43 (dt, J = 9.2, 7.6 Hz) 0 7-CH 5.06 (d, J = 9.5 Hz) 5.07 (d, J = 9.6 Hz) CH α 4.11 (dd, J = 8.5, 7.0 Hz) 4.11 (dd, J = 8.4, 7.2 Hz) 0 15-CH β 3.91 (t, J = 9.3 Hz) 3.91 (dd, J = 9.6, 8.8 Hz) 0 3-CH 3.84 (m) 3.84 (m) 0 17-CH 3.59 (d, J = 9.0 Hz) 3.59 (d, J = 8.0 Hz) 0 21-Me 1.51 (s) 1.51 (s) 0 19-Me 0.92 (s) 0.91 (s) 0.01 Comparison of 13 C NMR chemical shifts (Py-d5) Synthetic Glaucogenin D Proton Natural Glaucogenin D 2 (100 MHz) Δδ C C C C C C C C C C C C C C C C C C C C C Dou, J.; Bi, Z.-M.; Zhang, Y.-Q.; Li, P. Chin. J. Nat. Med. 2006, 4, 192. S15

17 5. NMR Spectra: S16

18 1 H NMR (after exchange with D 2 O) 1 H NMR (original spectrum) S17

19 S18

20 COSY S19

21 NOESY S20

22 NOESY S21

23 S22

24 S23

25 DEPT 135 DEPT C NMR S24

26 S25

27 S26

28 S27

29 S28

30 DEPT 135 DEPT C NMR S29

31 S30

32 S31

33 S32

34 S33

35 S34

36 S35

37 DEPT 135 DEPT C NMR S36

38 S37

39 1 H NMR (after exchange with D 2 O) 1 H NMR (original spectrum) S38

40 S39

41 DEPT 135 DEPT C NMR S40

42 S41

43 S42

44 1 H NMR (after exchange with D 2 O) 1 H NMR (original spectrum) S43

45 S44

46 DEPT 135 DEPT C NMR S45

47 S46

48 S47

49 S48

50 DEPT 135 DEPT C NMR S49

51 S50

52 S51

53 DEPT 135 DEPT C NMR S52

54 S53

55 S54

56 DEPT 135 DEPT C NMR S55

57 S56

58 S57

59 S58

60 DEPT 135 DEPT C NMR S59

61 S60

62 S61

63 DEPT 135 DEPT C NMR S62

64 S63

65 grease S64

66 DEPT 135 DEPT 90 grease 13 C NMR S65

67 S66

68 S67

69 Glaucogenin D (100 MHz, Py-d5) S68