Three New Cytotoxic Steroidal Alkaloids from Sarcococca hookeriana

Three new steroidal alkaloids with an unusual 3α tigloylamide group, named sarchookloides A–C (1–3), were isolated along with four known compounds (4–7) from the roots of Sarcococca hookeriana. Their structures and relative configuration were elucidated on the basis of spectroscopic methods including MS, UV, IR, 1D, and 2D NMR data. The isolated compounds were evaluated for their cytotoxicity against five human cancer cell lines: Hela, A549, MCF-7, SW480, and CEM in vitro. All three amide substituted steroidal alkaloids exhibited significant cytotoxic activities with IC50 values of 1.05–31.83 μM.

The 13 C NMR data of the ring A, coupling constants of H-2, H-3 and H-4 and NOESY data clearly indicated that compound 1 differs from the previously reported compounds of hookerianamide M [10] and sarcovagine A [18] with respect to the stereochemistry at C-2, C-3, and C-4 positions. In the ROESY spectrum ( Figure 2), the correlations of H-19 [δH 1.21 (3H, s)] with OH-2 and OH-4 indicated the β orientations of the two hydroxyl groups. Furthermore, the obvious ROESY correlations ( Figure 2) of HN with H-2, H-4 and H-5 [δH 1.26 (1H, m)] implied the α orientation of the tigloylamide group. The ring A of compound 1 may exist mainly as a stable boat conformation due to the substitution of 3α tigloylamide group [17]. Consequently, the structure and relative configuration of compound 1 was determined as (20S)-20-N,N-dimethylamino-2β,4β-dihydroxyl-3α-tigloylamino-5α-pregnane, which was named sarchookloide A ( Figure 1). Compound 2 had a molecular formula of C28H48N2O2, which was determined by HR-ESI-MS (m/z 445.3775 [M + H] + ), suggesting six degrees of unsaturation. The IR spectrum displayed absorptions indicating a hydroxyl group (3443 cm -1 ), amide carbonyl group (1664 cm −1 ) and double bond (1629 cm −1 ). In the 13 C NMR and DEPT spectra (Table 1), 28 carbon signals were observed, including 23 carbon resonances assigned to a 20α-dimethylamino-3-amino-5α-pregnane type steroidal alkaloid skeleton and 5 carbon resonances for a tigloyl group [21]. The similarity of the NMR data of compounds 2 and 20α-dimethylamino-2α-hydroxyl-3β-tigloylamino-5α-pregnane [16] suggested that they possessed the same planar structure. The ROESY correlations (Figure 2) of HN with H-2 and H-5 [δH 1.07 (1H, m)] implied the α orientation of the tigloylamide group and the β orientation of the hydroxyl group. This was also supported by the discrepant W1/2 (14.8) of H-2 [10] and the 13 C NMR data of the ring A in 2 compared with the data of reported compounds [16]. The substitution of 3α tigloylamide group led to the main boat conformation of the ring A in compound 2. Consequently, the structure and relative configuration of compound 2 was determined as (20S)-20-N,N-dimethylamino-2β-hydroxyl-3α-tigloylamino-5α-pregnane, which was named sarchookloide B (Figure 1  The similarity of the NMR data of compounds 2 and 20α-dimethylamino-2α-hydroxyl-3β-tigloylamino-5α-pregnane [16] suggested that they possessed the same planar structure. The ROESY correlations (Figure 2) of HN with H-2 and H-5 [δ H 1.07 (1H, m)] implied the α orientation of the tigloylamide group and the β orientation of the hydroxyl group. This was also supported by the discrepant W 1/2 (14.8) of H-2 [10] and the 13 C NMR data of the ring A in 2 compared with the data of reported compounds [16]. The substitution of 3α tigloylamide group led to the main boat conformation of the ring A in compound 2. Consequently, the structure and relative configuration of compound 2 was determined as (20S)-20-N,N-dimethylamino-2β-hydroxyl-3α-tigloylamino-5α-pregnane, which was named sarchookloide B (Figure 1).
Compound 3 was given the molecular formula C 28 H 48 N 2 O according to its HR-ESI-MS data at m/z 429.3829 [M + H] + (calculated as 429.3839), corresponding to six degrees of unsaturation. The IR spectrum of compound 3 included the absorption bands for amide carbonyl group (1665 cm −1 ) and double bond (1625 cm −1 ). The 13 C NMR and DEPT spectra (Table 1) of compound 3 exhibited 28 carbon signals corresponding to four quaternary carbons, eight methines, nine methylene, and seven methyl groups, of which 23 carbon resonances were ascribed to 20α-dimethylamino-3-amino-5α-pregnane type steroidal alkaloid skeleton and five carbon resonances were attributed to a tigloyl group [21].
Compound 3 and pachysamine G [19] exhibited the same planar structure, which was supported by the similar NMR data found for both of them. The α-orientations of the tigloylamide group was assigned by the ROESY correlations (Figure 2) of HN [δ H 5.93 (1H, d, J = 6.6 Hz)] with H-5 [δ H 1.11 (1H, m)] in combination with the different shift of the ring A in compound 3 compared with the data of pachysamine G in 13 C NMR spectrum. Similar to compounds 1 and 2, the boat conformation of the ring A in compound 3 was the main conformation [17]. Therefore, the structure and relative configuration of compound 3 was determined as (20S)-20-N,N-dimethylamino-3α-tigloylamino-5α-pregnane, which was named sarchookloide C ( Figure 1).
Compound 3 and pachysamine G [19] exhibited the same planar structure, which was supported by the similar NMR data found for both of them. The α-orientations of the tigloylamide group was assigned by the ROESY correlations (Figure 2) of HN [δH 5.93 (1H, d, J = 6.6 Hz)] with H-5 [δH 1.11 (1H, m)] in combination with the different shift of the ring A in compound 3 compared with the data of pachysamine G in 13 C NMR spectrum. Similar to compounds 1 and 2, the boat conformation of the ring A in compound 3 was the main conformation [17]. Therefore, the structure and relative configuration of compound 3 was determined as (20S)-20-N,N-dimethylamino-3α-tigloylamino-5α-pregnane, which was named sarchookloide C ( Figure 1).
The known compounds 4-7 were identified as pachysamine G (4) [19], pachysamine H (5) [19], sarcovagine B (6) [18], and pachyaximine A (7) [20] through a comparison of their spectroscopic data with those reported in the literature.    Table 2. Compound 5 had the greatest cytotoxicity to all cells, as its range of IC 50 values was approximately 1.05-2.23 µM. All three amide-substituted compounds, except for pachyaximine A, exhibited significant cytotoxic activity on all cells, which suggests that the amide group of these compounds was the necessary group for the cytotoxicity. In addition, Hela and A549 were the more sensitive cell lines to these types of compounds compared to all tested cancer cells because the IC 50 values of all compounds were less than 10 µM. Furthermore, all active compounds showed effects that were comparable to the chemotherapeutic drug adriamycin in inhibiting the growth of all cancer cells, which suggests that three amide-substituted pregnane-type steroidal alkaloids might have the potential to be anticancer agents.

General Experimental Procedures
Optical rotations were obtained on a JASCO model 1020 polarimeter (Horiba, Tokyo, Japan). UV spectra were measured on a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Kyoto, Japan). IR (KBr) spectra were measured on a Bio-Rad FTS-135 spectrometer (Bio-Rad, Hercules, CA, USA). The 1D and 2D NMR spectra were recorded on Bruker AVANCE III-600 spectrometers with TMS used as an internal standard (Bruker, Bremerhaven, Germany). The mass spectra were obtained on a Waters AutoSpec Premier P776 (Waters, NY, USA). The silica gel (200-300 mesh) for column chromatography and the TLC plates (GF 254 ) were obtained from Qingdao Marine Chemical Factory (Qingdao, Shandong, China). The Sephadex LH-20 (20-150 µm) used for chromatography was purchased from Pharmacia Fine Chemical Co. Ltd. (Pharmacia, Uppsala, Sweden). Fractions were visualized by heating silica gel plates sprayed with Dragendorff's reagent. The cell lines Hela, A549, MCF-7, SW480, and CEM were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), while MTT were obtained from Sigma Company.

Cytotoxicity Assay
The cytotoxicity of compounds 1-7 was tested on the human cervical cancer cell line Hela, lung adenocarcinoma cell line A549, breast cancer cell line MCF-7, colon cancer cell line SW480 and leukemia CEM cells. All cells were cultured in a RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), which was supplemented with 10% fetal bovine serum (Hyclone) in 5% CO 2 at 37 • C. The cytotoxicity assay was performed using the MTT method in 96-well microplates [22]. Briefly, the adherent cells (100 µL) were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before the addition of the drug. The suspended cells were seeded just before the addition of the drug at an initial density of 1 × 10 5 cells/mL. Each tumor cell line was exposed to the tested compound at different concentrations for 48 h. The experiments were performed in triplicate. Adriamycin (Sigma, St. Louis, MO, USA) was used as a positive control. After treatment, cell viability was measured and the cell growth curve was plotted. The IC 50 values were calculated by the Reed and Muench method [23].