Cytotoxic polyhydroxylated pregnane glycosides from Cissampelos pareira var. hirsuta

Fourteen new polyhydroxylated pregnane glycosides, cissasteroid A–N (1–14), and five known analogues (15–19), were isolated from the dried whole plant of Cissampelos pareira var. hirsuta. Their structures and stereochemistry were elucidated by extensive spectroscopic data, chemical hydrolysis, and ECD measurements. All the compounds were tested for their cytotoxicity against five human cancer cell lines, and inhibitory activity against NO release in LPS-induced RAW 264.7 cells. Compared with cisplatin, compound 7 showed more potent cytotoxicities against the HL-60, A549, SMMC-7721, MCF-7, and SW480 cell lines, with IC50 values of 2.19, 14.38, 2.00, 7.58, and 7.44 μM, respectively. The preliminary study of structure–activity relationship indicated that benzoic acid esterification at C-20 may have a negative effect on the cytotoxic activity of polyhydroxylated pregnane derivatives in these five human cancer cell lines. These results revealed the potential of compound 7 as an ideal antitumor lead compound.

Compound 3 was obtained as a white amorphous powder. Its 1 H and 13 C NMR data (Tables 1 and 2 Compound 4 was obtained as a white amorphous powder. Its 1 H and 13 C NMR spectra (Tables 1 and 2) bore a resemblance to those of 1, with the notable difference given by the presence of one additional b-cymaropyranosyl group [the anomeric proton d H 4.77 (1H, dd, J ¼ 9.6, 1.7 Hz, H-1 000 ), seven carbon signals d C 101.2 (C-1 000 ), 36.7 (C-2 000 ), 78.6 (C-3 000 ), 74.5 (C-4 000 ), 71.3 (C-5 000 ), 18.7 (C-6 000 ), 59.3 (C-7 000 )] in 4. Acid hydrolysis of 4 yielded only Dcymaropyranose. The HMBC cross peaks of H-1 00 (d H 4.85) with C-3 (d C 79.4), and H-1 000 (d H 4.77) with C-4 00 (d C 83.8), indicated that one cymaropyranosyl was at C-3 of the aglycone and the other was substituted at C-4 00 of the inner cymarose. Therefore, compound 4 was identied as 20-O-trans-cinnamoylsarcostin 3- and named cissasteroid D.  3.41, s  Compound 5 was obtained as a white amorphous powder. It gave the same molecular formula C 44 H 64 O 13 as that of 4, based on a sodium adduct ion m/z 823.4244 (calcd 823.4245). A comparison of the NMR spectroscopic data demonstrated that the difference between these two compounds was in the linkage position of the cinnamoyl group. The HMBC correlation from H-12 (d H 4.77) to C-9 0 (d C 168.4) conrmed that the cinnamoyl group was located at C-12. From the above analysis, compound 5 was characterized as 12-O-trans-cinnamoylsarcostin

Conclusions
Compounds 1-19 represent the rst report of polyhydroxylated pregnane glycosides from the genus Cissampelos. This also lays a solid chemical foundation for pharmacological research of C. pareira var. hirsuta. Compound 7 was the most promising of all isolated compounds based upon their IC 50 values. Further studies are necessary to explore antitumor mechanism, cytotoxicities in normal cells, and structure optimization.

General experimental procedures
Optical rotations and ECD spectra were measured by a Rudolph AP-IV polarimeter (Rudolph, Hackettstown, NJ, USA) and an Applied Photophysics Chirascanq CD spectropolarimeter (Applied Photophysics, Leatherhead, Surrey, UK), respectively. UV and IR spectra were acquired using a ThermoEVO 300 spectrometer (Thermo, Waltham, MA, USA) and a ThermoNicolet IS 10 spectrometer (Thermo, Waltham, MA, USA), respectively. NMR and mass spectra were recorded on a Bruker Avance III 500 spectrometer (Bruker, Germany) and a Bruker maXisHD mass spectrometer (Bruker, Germany), respectively. Preparative HPLC separations were performed on a SEP system (Beijing Sepuruisi scientic Co., Ltd, China) equipped with a variable-wavelength UV detector, using a YMC-Pack ODS-A column (250 Â 20 mm, 5 mm). Monosaccharide isolation was conducted on a Waters 2695 separation module with an evaporative light scattering detector (ELSD) (Waters, Milford, MA, USA). MCI gel CHP-20, ODS gel (50 mm), sephadex LH-20 (40-70 mm), and silica gel (160-200 mesh) were acquired from TOSOH Corp., Tokyo, Japan, YMC Group, Kyoto, Japan, Amersham Pharmacia Biotech AB, Uppsala, Sweden, and Marine Chemical Industry, Qingdao, China, respectively. Chemical reagents for isolation were of analytical grade and purchased from Tianjin Siyou Co., Ltd, China. Biological reagents were from Sigma Company. Human hepatocellular carcinoma (SMMC-7721) cell line was bought from China Infrastructure of Cell Line Resources (Beijing, China), from Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, China. Human myeloid leukemia cell line (HL-60), lung cancer (A549), breast cancer (MCF-7), and colon cancer (SW-480) were from American Type Culture Collection (ATCC, Manassas, VA, USA).

Plant material
The dried whole plants of C. pareira var. hirsuta were collected in Yunnan province, China, in July 2018, and authenticated by Prof. Cheng-Ming Dong at School of Pharmacy, Henan University of Chinese Medicine, where a voucher specimen (SE 20180705) was deposited.

Absolute conguration determination of sugar moieties
A solution of 10 (2.2 mg) in 1 mL of MeOH was hydrolyzed with 100 mL of 0.05 N H 2 SO 4 . The solution was stirred at 60 C for 2 h. Aer cooling, the reation mixture was diluted with 10 mL of H 2 O and extracted with 10 mL of CH 2 Cl 2 . The H 2 O phase was neutralized with saturated aqueous Ba(OH) 2 solution. The precipitate was ltered off, and then the ltrate was evaporated under reduced pressure to give the sugar fraction. The residue was isolated by preparative HPLC-ELSD (a TSKgel G3000PWXL column, 300 mm Â 7.8 mm, 5 mm) and eluted with H 2 O at a ow of 0.2 mL min À1 to obtain oleandropyranose (t R 53.9 min) and cymaropyranose (t R 55.3 min), respectively. Compounds 1-9, and 11-14 were hydrolyzed by the above procedure. D-Oleandrose and D-cymarose were identied by comparing their experimental and reported rotation values. 16 Cytotoxicity assay in vitro Cytotoxicity was tested by the MTS method previously described. 25 Cisplatin and paclitaxel were used as positive control. The cytotoxicity of compounds 1-19 was evaluated against HL-60, A-549, SMMC-7721, MCF-7, and SW-480 cell lines. All the cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum at 37 C in a humidied 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-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). To be brief, 100 mL of adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 24 h before drug addition, each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h. Aer the incubation, MTS (20 mL) was added to each well, and the incubation continued for 4 h at 37 C. The optical density of each well was measured at 492 nm in a 96-well microtiter plate reader. The IC 50 value of each compound was calculated by the Reed-Muench's method.

NO inhibitory activity
The NO inhibitory activity was evaluated by the previously reported protocol. 25 L-N G -Monomethyl arginine (L-NMMA) was used as a positive control. RAW 264.7 macrophages cells (2 Â 10 5 cells per well) were precultured in 96-well microplates for 24 h. The test compounds (50 mM) and L-NMMA with 1 mg mL À1 LPS were added and incubated for another 18 h at 37 C. Nitric oxide production was assessed by the Griess Reagent System.