Three New Highly Oxygenated Germacranolides from Carpesium Divaricatum and Their Cytotoxic Activity

Three new highly oxygenated (2–4), and two known (1 and 5) germacranolides, were isolated from the whole plant of Carpesium divaricatum. The planar structures and relative configurations of the new compounds were determined by detailed spectroscopic analysis. The absolute configuration of 1 was established using the circular dichroism (CD) method and X-ray diffraction, and the stereochemistry of the new compounds 2–4 were determined using similar CD spectra with 1. The new compound 2 and the known compound 5 exhibited potent cytotoxicity against hepatocellular cancer (Hep G2) and human cervical cancer (HeLa) cells, superior to those of the positive control cis-platin.

As a part of our ongoing search for new bioactive products from medicinal plants in China, three new, and two known highly oxygenated germacranolides representing other subtype (Figure 1), As a part of our ongoing search for new bioactive products from medicinal plants in China, three new, and two known highly oxygenated germacranolides representing other subtype (Figure 1), were isolated from the whole plant of C. divaricatum. In this paper, a structural elucidation and bioactive evaluation of these compounds is presented.

Purification of Compounds 1-5
The whole plant of C. divaricatum was extracted three times with EtOH-H 2 O (95:5). The five highly oxygenated germacranolides were isolated and purified via silica gel chromatograpy, Sephadex LH-20 gel chromatograpy, and semi-preparative High-Performance Liquid Chromatography (HPLC).
Compounds 3-4 possessed molecular formulas of C23H32O9 and C25H38O9, from their HRESIMS at m/z 475.1939 [M + Na] + and m/z 505.2414 [M + Na] + respectively. The 1 H and 13 C NMR data of 3-4 were similar to those of 1, except that the 2-methacryloyloxy group at C-9 in 3 was observed in place of 3-methylbutyryloxy group in 1, and an isobutyryloxy group at C-5 and the 3-methylbutyryloxy group at C-9 in 1 were replaced by two 2-methylbutyryloxy groups in 4, respectively. These observations were confirmed by analyses of relevant 1 H-1 H COSY, HSQC and HMBC data ( Table 1). The relative configurations of 3-4 were determined to be the same as those of 1, by comparison of ROESY data for relevant protons. Similar CD data of 3-4 and 1 (Figure 2) revealed the same absolute configurations of 3-4 as that of 1. Thus, the structures of compounds 3-4 were established as shown, and named divarolide F and divarolide G respectively.
The structure of the known compound (5) was identified as 4β,8α-dihydroxy-5β-2methylbutyryloxy-9β-3-methylbutyryloxy-3-oxo-germacran-7β,12α-olide [5], by comparison of its spectroscopic data with reported data. were similar to those of 1, except that the 2-methacryloyloxy group at C-9 in 3 was observed in place of 3-methylbutyryloxy group in 1, and an isobutyryloxy group at C-5 and the 3-methylbutyryloxy group at C-9 in 1 were replaced by two 2-methylbutyryloxy groups in 4, respectively. These observations were confirmed by analyses of relevant 1 H-1 H COSY, HSQC and HMBC data ( Table 1). The relative configurations of 3-4 were determined to be the same as those of 1, by comparison of ROESY data for relevant protons. Similar CD data of 3-4 and 1 (Figure 2) revealed the same absolute configurations of 3-4 as that of 1. Thus, the structures of compounds 3-4 were established as shown, and named divarolide F and divarolide G respectively.

X-ray Crystal Structure Analysis of Compound 1
X-ray diffraction data were collected on the Agilent GEMINI TM E instrument (CrysAlisPro software, Version 1.171.35.11), with enhanced Cu Kα radiation (λ = 1.54184 Å). The structure was solved by direct methods and refined by full-matrix least-squares techniques (SHELXL-97). All non-hydrogen atoms were refined with anisotropic thermal parameters. Hydrogen atoms were located by geometrical calculations and from positions in the electron density maps. Crystallographic data (excluding structure factors) for 1 in this paper has been deposited with the Cambridge Crystallographic Data Centre (deposition number CCDC 1570798). Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-12-23336033 or e-mail: deposit@ccdc.cam.ac.uk).  Cell viability assay: The assay was run in triplicate. In a 96-well plate, each well was plated with 2 × 10 4 cells. After cell attachment overnight, the medium was removed, and each well was treated with 100 µL of medium containing 0.1% DMSO or different concentrations of the test compounds and the positive control cis-platin. The plate was incubated at 37 • C for 4 days in a humidified, 5% CO 2 atmosphere. Cytotoxicity was determined using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay [28]. After the addition of a 10 µL MTT solution (5 mg/mL), cells were incubated at 37 • C for 4 h. After adding 150 µL DMSO, cells were shaken to mix thoroughly. The absorbance of each well was measured at 540 nm in a Multiscan photometer. The IC 50 values were calculated by Origin software and listed in Table 2.
Statistical analysis: Values were expressed as mean ± SD. Statistical analyses were performed using the Student's t-test. Differences were considered significant when associated with a probability of 5% or less (p ≤ 0.05).

Conclusions
In conclusion, three new compounds (2-4), as well as two known compounds (1 and 5), were isolated from the whole plant of C. divaricatum. Structurally, all compounds contained a 5-membered γ-lactone ring fused to a circular 10-membered carbocycle. We obtained a set of isomers (2/4/5) from the same plant. The isolation of these isomers is a huge challenge because they are highly oxygenated and have similar structures. The absolute configuration of compound 1 was unambiguously established by X-ray diffraction. The other compounds with the same skeleton were determined by comparison of NOESY and CD data with those of 1. Compounds 1 and 5 showed significant cytotoxicity against two human tumor cell lines. These findings are an important addition to the present knowledge on the structurally diverse and biologically significant germacranolide family.