Tuberostemonine O from the Roots of Stemona tuberosa

Experimental

General Experimental Procedures. Optical rotation was measured on a P-1010 polarimeter (JASCO, Japan) at 27 °C. IR spectrum was recorded on Nicolet iS10 FT-IR spectro-meter (Thermo Fisher Scientific, MA). 1D and 2D NMR experiments were performed on a UNITY INOVA 400 MHz FT-NMR instrument (Varian, CA) with tetramethylsilane (TMS) as internal standard. HREIMS was performed with Waters ACQUITY UPLC system coupled to a Micromass Q-Tof Micro mass spectrometer and Agilent 6220 Accurate-Mass TOF LC/MS system. Silica gel (70-230 mesh and 230-400 mesh, Merck, Germany) was used for column chromato-graphy (CC). Thin-layer chromatographic (TLC) analysis was performed on Kieselgel 60 F 254 plates (silica gel, 0.25 mm layer thickness, Merck, Germany), with visualization under UV light (254 and 365 nm) and Dragendorff’s reagent.

Figure 3.Key NOESY correlations of 1. The Energy minimized stereostructure of 1 was constructed by MM2 calculation using CAChe 5.0™ molecular modeling program.

Plant Material. The dried roots of S. tuberosa were purchased from the Insan Oriental Herbal Market in Seoul, Korea. The sample was identified by Prof. Je-Hyun Lee (College of Oriental Medicine, Dongguk University, Korea). A voucher specimen (No. EA322) has been deposited at the Natural Product Chemistry Laboratory, College of Pharmacy, Ewha Womans University, Korea.

Extraction and Isolation. The roots of S. tuberosa (10 kg) were extracted with MeOH (3 × 15 L, 24 h) at room temperature. The solvent was evaporated in vacuo to afford a MeOH extract (3.6 kg), which was then suspended in distilled water, and partitioned with n-hexane (3 × 5 L), EtOAc (6 × 5 L), and n-BuOH (6 × 5 L), successively. The EtOAc-soluble extract (98 g) was separated by silica gel CC (φ 10 cm; 70-230 mesh), using gradient mixtures of CH2Cl2-MeOH (100:0→0:100) as mobile phases, affording 12 fractions (F1-F12). The fraction F6 (3.4 g) was subjected to silica gel CC (φ 3 cm; 230-400 mesh) and eluted with gradient solvent systems of n-hexane-acetone (5:1→1:1) to yield compound 1 (21.8 mg, 0.00023% w/w). The fraction F10 (11.9 g) was chromatographed on silica gel (φ 3 cm; 230-400 mesh) with gradient mixtures of n-hexane-acetone (9:1→2:3) to give tuberostemonine N (3.8 g, 0.04% w/w).

Tuberostemoine O (1): Light yellow powder. (c 0.09, CHCl3); IR (KBr) νmax cm−1: 2924, 1770, 1457, 1189, 1018; 1H- and 13C-NMR data, see Table 1; HRESIMS (positive mode) m/z = 376.2483 [M+H]+ (calcd for C22H34NO4: 376.2482).