General Proceduces. MS were determined on an API Qstar Pulsa LC/TOF mass spectrometer. NMR spectra were measured on a Bruker DRX-500 spectrometer with TMS as internal standard. Silica gel (200-300 mesh) was used for column chromatography and silica gel GF254 for TLC (Qingdao Marine Chemical Co., China). Solvents were of industrial purity and distilled prior to use.
Plant materials. The whole plant of Bulbophyllum odoratissimum were collected from Simao County of Yunnan Province, China in February, 2004 and identified by one of the authors, Dr. Hong Wang, School of Life Science, Yunnan University, where a voucher specimen (No.0402017) was deposited.
Extraction and isolation. The air-dried powdered whole plant of B. odoratissimum (20 Kg) were extracted with 95% EtOH (20 liters × 4) at room temperature. The EtOH extract was concentrated in vacuo to yield a dark brown residue (1 Kg). H2O (2.5 L) was added to the residue, and the resultant solution was extracted with petroleum ether, EtOAc and n-BuOH successively (1.5 liters ×4). The EtOAc extract (350 g) was applied to a silica gel column, eluting with petroleum ether containing increasing amounts of acetone to obtain 6 fractions. Fr. 3 (77 g) was separated to two subfractions by silica gel column chromatography (petroleum ether-acetone 4:1, 7:3). The second subfraction (47 g) was subjected to repeated column chromatography, first on silica gel (CHCl3-acetone 80:1) and then on Sephadex LH-20 (MeOH-H2O 9:1) to obtain 1 (21 mg).
Compound 1: 1H-NMR (500 MHz, CD3OD) and 13C-NMR (125 MHz, CD3OD), see Table 1; EI-MS m/z (70 eV, ret. Int., %): 300 [M+] (100), 285 (63), 253 (17), 242 (22), 214 (33), 185 (11), 150 (16); HRESIMS m/z: 323.0889 [M+Na]+, requires 323.0895.
Methylation of 1: compound 1 (10 mg) was methylated with CH2N2 in Me2CO. Preparative TLC of the crude product (silica gel, petroleum ether-EtOAc 4:1) gave 2,3,4,6,7-pentamethoxyphenanthrene (1b, 6 mg) as a colorless amorphous powder. EI-MS m/z (70 eV, ret. Int., %): 328 [M+] (100); 1H-NMR (CD3COCD3): δ9.06 (1H, s, H-5), 7.61 (1H, d, J=9.0 Hz, H-9), 7.57 (1H, J=9.0 Hz, H-10), 7.34 (1H, s, H-1 or H-8), 7.27 (1H, s, H-8 or H-1), 4.04, 4.01, 3.99, 3.96, 3.96 (15H, each s, OMe ×5), identical to lit. values.4