schiglautone a, a new tricyclic triterpenoid with a unique

1

Schiglautone A, a New Tricyclic Triterpenoid with a Unique

6/7/9-Fused Skeleton from the Stems of Schisandra glaucescens

Fan-Yu Meng,† Jia-Xiang Sun,

† Xue Li,

† Heng-Yi Yu,

†

Shu-Ming Li, ‡ and Han-Li Ruan*

,†

†Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and

Technology, Hangkong Road 13, 430030, Wuhan, P. R. China; ‡ Institut of

Pharmaceutical Biology and Biotechnology, Philipps University of Marburg,

Deutschhausstrasse 17A, D-35037, Germany.

* Tel: 0086-27-83657870 E-mail: ruanhl@mails.tjmu.edu.cn

2

Content list:

S1. Experimental Section

S2. Crystal cell diagram for Schiglautone A (1)

S3. 1H NMR spectrum (400 MHz, C5D5N) of 1

S4. 13

C and DEPT spectra (101 MHz, CDCl3) of 1

S5. 1H-

1H COSY spectrum (400 MHz, C5D5N) of 1

S6. HSQC spectrum (400 MHz, C5D5N) of 1

S7. HMBC spectrum (400 MHz, C5D5N) of 1

S8. NOESY spectrum (400 MHz, C5D5N) of 1

S9. ESI-MS interpretation of 1

S10. HR-ESI-MS interpretation of 1

S11. IR spectrum of 1

S12. CD spectrum of 1

3

S1. Experimental Section

1.1 General Experimental Procedures.

IR spectrum was recorded with a PerkinElmer Spectrum one FT-IR spectrometer, as

KBr pellets. Specific optical rotations were obtained on a PE-341LC automatic

polarimeter. 1

H-, 13

C-, and 2D-NMR spectra were taken on a Bruker-AM-400

spectrometer, with TMS as internal standard. The CD spectrum was obtained with a Jasco

J-810 spectropolarimeter. The ESI-MS was measured with a Thermo Finnigan LCQ

DECA. XP plus. HR-ESI-MS was measured on Thermo Scientific LTQ-Orbitrap XL

mass spectrometer. MPLC was performed on BUCHI pump module C-605 apparatus

using BUCHI plastic-glas column (36mm×460mm). Silica gel (SiO2; 200–300, or

300-400 mesh, Qingdao Marine Chemical Inc., Qingdao, China), Sephadex LH-20 gel

(GE Healthcare) and MCI-gel (CHP20P, 75–150 mm, Mitsubishi Chemical Industries

Ltd.) were used for column chromatography. Fractions were monitored by TLC (SiO2;

Yan-tai Institute of Chemical Technology). Spots were visualized by heating silica gel

plates immersed in ethanol -H2SO4.

1.2 Plant Material.

The stems of Schisandra glaucescens were collected in the Shennongjia Mountain

areas of Hubei Province P. R. China, in November 2009, and identified by Mr. Shigui Shi

from Shennongjia Institute for Drug Control. A voucher specimen (ID 20091101) has

4

been deposited with Herbarium of Materia Medica, Faculty of Pharmacy, Tongji Medical

College of Huazhong University of Science and Technology, P. R. China.

1.3 Cellular Proliferation Assay

The cytotoxic activity of 1 was evaluated using human HeLa cell line, Hep G2 cell

line and gastric cancer cells (SGC-7901). All cells were cultured in DMEM medium

supplemented with 10% fetal calf serum (FCS), at 37 °C in a humidified atmosphere with

5% CO2. The viability of the cells was determined using the MTT colorimetric assay

system1. Briefly, 90µl cells were seeded into each well of a 96-well cell culture plate and

allowed to adhere for 12 h before drug addition, then, each tumor cell line was exposed

to the test compound at various concentrations in triplicate for 32 h, with 5-fluorouracil

(5-FU) as positive control. After the incubation, MTT (75 µg) was added to each well,

and the incubation continued for 4 h at 37 °C. 100µl of DMSO was added after the

supernatant was doffed off, and the percentage of viable cells was quantified at 570 nm

in an ELISA reader. For statistical analysis, the inhibitory rate of compound 1 and 5-FU

were calculated.

References

(1) Zheng, Y. T.; Ben, K. L.; Zhang, F.; Wang, J. H. Immunopharm. Immunot. 1995, 17,

69.

5

S2. Crystal cell diagram for Schiglautone A (1)

6

S3. 1H NMR spectrum (400 MHz, C5D5N) of 1

7

S4. 13

C and DEPT spectra (101 MHz, CDCl3) of 1

8

9

S5. 1H-

1H COSY spectrum (400 MHz, C5D5N) of 1

10

S6. HSQC spectrum (400 MHz, C5D5N) of 1

11

S7. HMBC spectrum (400 MHz, C5D5N) of 1

12

S8. NOESY spectrum (400 MHz, C5D5N) of 1

13

S9. ESI-MS interpretation of 1

14

S10. HR-ESI-MS interpretation of 1

15

S11. IR spectrum of 1

16

S12. CD spectrum of 1