Hypercohones D–G, New Polycyclic Polyprenylated Acylphloroglucinol Type Natural Products from Hypericum cohaerens

Graphical Abstract Four new polycyclic polyprenylated acylphloroglucinol type metabolites, hypercohones D–G (1–4), along with four known analogues (5–8), were isolated from the aerial parts of Hypericum cohaerens. 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. Electronic supplementary material The online version of this article (doi:10.1007/s13659-014-0007-5) contains supplementary material, which is available to authorized users.


Results and Discussion
The MeOH extract of the air-dried and powdered aerial parts of H. cohaerens (10.0 kg) was subjected to a silica gel column to afford five fractions A-E. Fraction B was subjected to a series of chromatographic methods, and led to the isolation of four new acylphloroglucinol derivatives, hypercohones D-G (1-4), together with four known ones, uralodin A (5), [17] uralodin B (6), [18] oxepahyperforin (7) [19], and tomoeone E (8) [20].
Further analysis of the NMR data of 1 with those of hyperforin revealed that they were structurally similar to each other except that the signals for the methylene at C-34 in hyperforin was replaced by an oxygenated methine (d C 80.9) in 1, [10,11] as evidenced by the H 2 -33/H-34/H-35 unit observed in the 1 H-1 H COSY spectrum (Fig. 1). The existence of the epoxy group between C-34 and C-2 (d C 170.4) can be fully confirmed by the 11 indices of hydrogen deficiency. In addition, the HMBC correlations from Me-32 (d H 1.08, 3H, s) to C-1 (d C 73.9), C-7 (d C 39.5), C-8 (d C 49.0), and C-33 (d C 37.3) confirmed the structures furthermore.
The relative configuration of compound 1 was determined on the basis of a ROESY experiment (Fig. 1). The The IR spectrum showed absorption bands at 3441 (hydroxyl) and 1724, 1663 cm -1 (carbonyl groups). Extensive analysis of the 1D NMR spectroscopic data (Tables 1 and 2) of 2 exhibited a close resemblance with oxepahyperforin (7) [19]. The differences in the 1D spectral data of 2 compared to 7 were that the chemical shifts of C-32 (d C 15.  (Tables 1 and 2) [19]. However, the signals for the isopropyl group in 7 were replaced by signals for a phenyl group in 3, which was confirmed by HMBC correlations from both H-12 (d H 7  Me-16 suggested that 4 had the same relative configurations as Sampsonol C at C-8, C-9, C-12, and C-13 (Fig. 3). Therefore, the structure of 4 was established as illustrated and named hypercohone G.
Compounds 1-8 were tested for in vitro inhibitory activities against HL-60, SMMC-7721, A549, MCF-7 and SW480 human tumor cell lines using the MTT method described previously [22]. 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)

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 IC 50 value of each compound was calculated by Reed and Muench's method [22].