New Dimeric and seco-Abietane Diterpenoids from Salvia wardii

Two dimeric abietane diterpenoids, salviwardins A and B (1 and 2), and a seco-abietane diterpenoid salviwardin C (3), along with five known analogues (4–8), were isolated from the roots of Salvia wardii. The structures of these isolates were elucidated by extensive spectroscopic methods. The inhibitory activities of these isolates against five human cancer cell lines in vitro were also tested.

Salviwardin A (1) was obtained as orange powder. Its molecular formula C 40 H 54 O 4 was established by its 13 C NMR and HREIMS (m/z 598.4029, [M] ? ) data, indicating 14 degrees of unsaturation. The IR absorption at 3440 and 1624 cm -1 implied the existence of hydroxyl and carbonyl groups. The 13 C and DEPT NMR (Table 1) spectroscopic data of 1 revealed 40 carbon signals, comprising fifteen quaternary carbons (one carbonyl, nine olefinic, and one oxygenated group), seven methines (one oxygenated and three olefinic ones), eight methylenes and ten methyls. The 1 H NMR (Table 2) spectrum of 1 showed the presence of two isopropyl groups and six singlet methyls. The 13 C and DEPT NMR spectroscopic data showed four noticeable quaternary signals for abietane diterpenoid at d C 40.1 (s, C-4), d C 46.7 (s, C-10), d C 33.8 (s, C-4 0 ), d C 39.3 (s, C-10 0 ) [19,20]. These evidences indicated that compound 1 should be a dimer of two abietane diterpenoids units.
The two units account for 13 degrees of unsaturation. Since the totally degrees of unsaturation were 14, the remained one degree of unsaturation should be ascribed to the linkage between the two units through C-11/C-5/C-6 to C-11 0 /C-12 0 to create an additional ring. The HMBC of H-6/C-12 0 confirmed the linkage of C-6/C-12 0 through an oxygen atom. In the 1 H NMR spectrum of 1, an obvious OH signal can be found at d H 7.50, and this OH group can be deduced to be attached at C-11 as evidenced by its HMBC correlations with C-9 (d C 124.7), C-11 (d C 144.1), and C-12 (d C 181.8). Then, the remained oxygenated quaternary at (C-5) and the oxygenated aromatic quaternary carbon (C-11 0 ) were deduced to linked through ether bridge.
In the NOESY spectrum ( Fig. 1 The molecular formula of salviwardin B (2) was determined to be C 40 H 52 O 4 from its 13 C NMR and HRESIMS spectral data, indicating one more unsaturation than 1. Comparison of their 1D and 2D NMR data indicated that the structures of 1 and 2 were similar to each other (Tables 1, 2). The difference lied in that the two methylenes (C-6 0 d C 19.3 and C-7 0 d C 32.4) in 1 were replaced by a double bond (C-6 0 d C 126.9 and C-7 0 d C 128.0) in 2, which indicated that 2 was a 6 0 ,7 0 -dehydrogen derivative of 1. This was confirmed by HMBC correlations from H-6 0 (d H 5.82) to C-4 0 (d C 33.3), C-5 0 (d C 51.6) and C-8 0 (d C 125.8), and from H-7 0 (d H 6.37) to C-5 0 and C-14 0 (d C 118.0). By detailed analysis of its ROESY (Fig. 2) spectrum, the relative configuration of 2 was also elucidated to be the same as that of 1. Ultimately, the structure of 2 was determined and named salviwardin B.    Fig. 3. Other parts of 3 were identical to those of the known compound by detailed analysis of the 1 H-1 H COSY and HMBC correlations (Fig. 3). Therefore, the structure of 3 was determined and named salviwardin C.
All isolates were tested for their in vitro inhibitory activities against HL-60, SMMC-7721, A549, MCF-7, and SW480 human tumor cell lines using the MTT method described previously [26]. The results indicated that all the compounds were inactive with IC 50 [ 30 lM.

General Experimental Procedures
Optical rotations were obtained with a Jasco P-1020 polarimeter. UV spectra were measured on Shimadzu UV-2401A spectraphotometer. IR spectra were detected on a Bruker Tensor-27 infrared spectrophotometer with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker AV-400, and Avance III-600 MHz spectrometers with TMS as the internal standard. Chemical shifts (d) were expressed in ppm with reference to the solvent signals. HRESIMS analysis and HREIMS were determined on API QSTAR time-of-flight spectrometer and on Waters Auto spec Premier P776 mass spectrometer. Semi-preparative HPLC was performed on an Agilent 1100 liquid chromatography with a Zorbax SB-C18 (9.4 mm 9 25 cm) column. Column chromatography was performed on Sephadex LH-20 (GE Healthcare), Silica gel (100-200 and 200-300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China), and Amphichroic RP-18 gel (40-63 lm, Merck, Darmstadt, Germany) and MCI gel (75-150 lm, Mitsubishi Chemical Corporation, Tokyo, Japan). Fractions were monitored by TLC and spots were visualized by heating silica gel plates sprayed with 10 % H 2 SO 4 in EtOH.

Plant Material
The roots parts of S. wardii were collected in Zuogong prefecture Tibet, China, in July 2011. The plant was

Cytotoxicity Assays
The following human tumor cell lines were used: HL-60, SMMC-7721, A-549, MCF-7, and SW-480, which were obtained from ATCC (Manassas, VA, USA). All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10 % fetal bovine serum (FBS, Hyclone) at 37°C in a humidified atmosphere with 5 % CO 2 . Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA). Briefly, 100 lL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before test compound addition, while suspended cells were seeded just before test compound addition, both with an initial density of 1 9 10 5 cells/mL in 100 lL of medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cis-platin and paclitaxel (Sigma) as positive control. After the incubation, MTT (100 lg) was added to each well, and the incubation continued for 4 h at 37°C. The cells were lysed with 100 lL of 20 % SDS -50 % DMF after removal of 100 lL of medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by Reed and Muench's method [25].