New Highly Oxygenated Germacranolides from Carpesium divaricatum and their Cytotoxic Activity

Eight highly oxygenated germacranolides (1–8) including four new ones (2–5) were isolated from the whole plant of Carpesium divaricatum. The planar structures and relative configurations of the new compounds were determined by NMR experiment and HRESIMS data. The absolute configuration of 1 was established by circular dichroism (CD) method and X-ray diffraction, and the stereochemistry of the new compounds 2–5 were determined by similar CD spectra with 1. Compound 2 is the first hydroperoxyl germacrane from the genus Carpesium. The 13C NMR data of 1, NMR data of 6–7, and their absolute configurations were reported for the first time. Two new compounds (2 and 4) and two known compounds (6 and 8) exhibited potent cytotoxicity against human cervical cancer (HeLa) cells, superior to that of the positive control doxorubicin.

(1H, d, J = 2.0 Hz, Hb-13), δ C 134.7 (C-11), 127.4 (C-13) and 170.6 (C-12); three carbonyl carbons at δ C 213.5 (C-9), 175.0 (C-1′ ) and 167.1 (C-1″ ); an oxygenated quaternary carbons at δ C 73.9 (C-4); five methines including three oxygenated ones at δ  13 C NMR data) of 2 were similar to those of 8 12 (Table S8.  Information), except for the ester residue at C-5. The singlet signals of 3′ -Me and 4′ -Me together with chemical shift difference of C-2′ (δ 34.9 → 84.8) implied that C-2′ was an oxygenated quaternary carbon compared with 8. Considering the chemical shift value of C-2′ (δ 84.8) and molecular formula of 2 confirmed that a hydroperoxyl moiety was attached at C-2′ [25][26][27] , which was further confirmed by the HRESIMS with fragment peaks at m/z 345. 1645 (Fig. 4) showed two partial structure sequences for 2: CH 2 (3)CH 2 (2)CH 2 (1)CH(10)CH 3 (14) and CH(5)CH(6)CH(7)CH (8). The C-C interconnectivity of all fragments was established from the HMBC spectrum (Fig. 4) as correlations of H-15 with C-3 and C-5, H-14 with C-1 and C-9, H-13 with C-7 and C-12, H-8 with C-1″ (ester carbonyl of angeloyloxy group), and H-5 with C-1′ (ester carbonyl of 2′ -hydroperoxyl-isobutyryloxy group). On the basis of these data, the planar structure of 2 was established.   The relative configuration of 2 was determined by analysis of ROESY data. The key NOE correlations of H-8/H-6, H-7/H-10, H-7/H-5 and H-5/H 3 -15 indicated that 2 had the same relative configuration as 1. The CD spectrum of 2 showed two positive Cotton effects at near 252 and 294 nm, which closely resembled those of 1. Similar ROESY and CD data of 2 and 1 assigned the absolute configuration of 2 as 4S, 5R, 6S, 7S, 8R and 10R. Thus, the structure of compound 2 was defined as shown, named divarolide A.  24 , except that the 2′ -hydroxy-isobutyryloxy group at C-5 and the angeloyloxy group at C-8 in 3 were observed in place of two isobutyryloxy groups in 1, and an isobutyryloxy group at C-8 in 1 was replaced by the 2-methylacryloyl group in 4, respectively. These observations were confirmed by analyses of relevant 1 H-1 H COSY, HSQC and HMBC data (Fig. 4). The relative configurations of 3-4 were determined to be the same as that of 1 by comparison of ROESY data for relevant protons. Similar CD data of 3-4 and 1 revealed the same absolute configurations of 3-4 as that of 1. Thus, the structures of compounds 3-4 were established as shown, named divarolide B and divarolide C, respectively.
The molecular formula of compound 5 was assigned as C 24 H 32 O 8 by HRESIMS (471.1988 [M + Na] + ). A comparison of the NMR data of 5 with those of 8 suggested that both of them had the same substituted groups at C-5 and C-8, but that the two mutually coupled methylene units (C-2-C-3) in 8 were oxidized to an olefin moiety in 5. The C-2/C-3 double bond was assigned E-geometry on the basis of the large coupling constant observed for olefinic protons (17.0 Hz). The H-1 H COSY, HSQC and HMBC spectra (Fig. 4) of 5 confirmed this observation, leading to the assignment of its planar structure. The relative and absolute configurations of 5 were deduced to be the same as those of 1, on the basis of similar ROESY and CD data. Thus, the structure of compound 5 was elucidated as shown, named divarolide D.
Compounds 6-7 shared the same molecular formula C 24 13 C NMR data of 6-7 showed a great similarity with those of 1, except for the ester residues at C-8. The isobutyryloxy group at C-8 in 1 was placed by a 3-methylbutyry-loxy group in 6 and the 2-methylbutyryloxy group of 7, respectively. Compounds 6-7 have been reported as a mixture from Inula cuspidata 24 . Actually, the exact linkage sites of the substituted groups have not been determined in the previous report and the authors speculate the mixture may contain two pairs of mixtures (incaspitolide B and C). Although the isolation of 6-7 is a huge challenge as they are highly oxygenated and similar, both of them were separated successfully in the present paper. Similarly, their relative and absolute configurations were determined as same as those of 1 by comparison of the ROESY and CD data. Thus, the structures of compounds 6-7 were established as shown, named incaspitolide B 1 and incaspitolide B 2 , respectively.
Compound 8 was a known analogue of 1-7, identified as (4S, 5R, 6S, 7S, 8R, 10R)-8-angeloyloxy-4-hydroxy-5-isobutyryloxy-9-oxo-germacran-7, 12-olide, by comparison of its MS, NMR and optical rotation data with reported data 12 .  In conclusion, eight highly oxygenated germacranolides including four new ones (2-5) were isolated from the whole plant of C. divaricatum. To the best of our knowledge, this is the first report of hydroperoxyl germacrane from the genus Carpesium. New compounds 2 and 4, as well as known compounds 6 and 8, exhibited potent cytotoxicity against HeLa cell lines, superior to that of the positive control doxorubicin. These findings are an important addition to the present knowledge on the structurally diverse and biologically important germacranolide family.
Divarolide C (4) X-ray crystal structure analysis. 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 1441395). 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).
A colorless triclinic crystal (0.50 × 0.50 × 0.40 mm) of 1 was obtained from CH 2 Cl 2 -MeOH (3:1). Crystal data: 3C 23  Cytotoxicity assays. 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 μ L of medium containing 0.1% DMSO or different concentrations of the test compounds and the positive control doxorubicin. The plate was incubated for 4 days at 37 °C 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 addition of 10 μ L MTT solution (5 mg/mL), cells were incubated at 37 °C for 4 h. After adding 150 μ L DMSO, cells were shaken to mix thoroughly. The absorbance of each well was measured at 490 nm in a Multiscan photometer. The IC 50 values were calculated by SPSS software and listed in Table 3.