Isolation, Structure Elucidation, and Absolute Configuration of Germacrane Isomers from Carpesium divaricatum

Five sets of germacrane isomers (1/8/17, 2/7/10/11/13/16/18, 3/4/5/14/20, 6/12/15, and 9/19) with different skeletal types, including seven new ones (1–3, 8–9, and 15–16) were isolated from the whole plant of Carpesium divaricatum. Among them, there are six pairs of stereoisomers (1/8, 2/13, 4/14, 6/12, 7/11 and 10/11). The planar structures and relative configurations of the new compounds were elucidated by detailed spectroscopic analysis. The absolute configurations of 4, 10, 11, and 17 were established by circular dichroism (CD) spectra and X-ray crystallographic analyses, and the stereochemistry of the new compounds 1–3, 8–9, and 15–16 were determined by similar CD spectra with 4, 10, 11, and 17, respectively. The confusion in the literature about subtypes I and II of germacranolides was clarified in this paper. The NMR data of 10–11, and the absolute configurations of the known compounds 4–6, 13–14, and 17–20 were reported for the first time. Compounds 13, 17, and 18 showed cytotoxicity against human cervical (HeLa), colon (LoVo) and stomach cancer (BGC-823) cell lines with IC50 values in the range 4.72–13.68 μM compared with the control cis-platin (7.90–15.34 μM).

contains a 5-membered α-methylene-γ-lactone ring linkage at C-7 and C-8, and the 2β,5β-hemiacetal group on the macro-ring system. Compounds 8-14 and the known compounds divaricin A-C 8 are assigned as subtype II (named 2α,5α-epoxygermacranolide), having a 7,8-α-methylene-γ-lactone ring and the 2α,5α-hemiacetal group. The known compounds incaspitolide A-C 22,23 and eight germacranolides we previously reported from C. divaricatum 12 are classified as subtype III (named 9-oxo-germacranolide) with structural features of one 6,7-α-methylene-γ-lactone ring and the 9-ketone group. Compounds 15-20 represent subtype IV (named 3-oxo-germacranolide), possessing a 6,7-α-methylene-γ-lactone ring and the 9-ketone group. Both subtypes I and II are 2,5-hemiacetal-linked germacranolides, but their configurations at the bridgehead carbons between 5-membered ring and 9-membered ring are opposite. Subtypes III and IV have similar skeletons, but linkage sites of the ketone group are different (9-ketone group in subtype III and 3-ketone group in subtype IV). Twenty analogues here we further isolated from same species represent other three subtypes of germacranolides (subtypes I-II and IV). NOESY spectrum, circular dichroism (CD) method and X-ray data analysis were used to confirm their relative and absolute configurations. In this paper, the isolation, the structural elucidation, the absolute configuration and bioactive evaluation of these compounds were present. The confusion in the literature about subtypes I and II of germacranolides is also discussed.

Results and Discussion
Structural Elucidation of Compounds from Subtype I. Compound 1 (Fig. 2) was obtained as white needles. The molecular formula was assigned as C 23 H 34 O 9 on the basis of the positive-ion HRESIMS ion at m/z 477.2104 [M + Na] + , together with its 1 H and 13 C NMR data (Tables 1 and 2). The IR spectrum showed the presence of hydroxyl (3493 cm −1 ) and carbonyl (1767 and 1737 cm −1 ) functional groups. The 1 H NMR spectrum of 1 displayed the presence of two isobutyryloxy groups, which was further confirmed by the HRESIMS data of  3 -15). These observations and analyses of 1 H-1 H COSY, HSQC, and HMBC spectra ( Fig. 3) suggested that the structure of 1 was similar to that of 2β,5-epoxy-5,10-dihydroxy-6α-angeloyloxy-9βisobutyryloxy-germacran-8α,12-olide (4) 9 , except for an isobutyryloxy group in 1 compared to the angeloyloxy group at C-6 in 4.
The relative configuration of 1 was determined by analysis of the NOESY data (Fig. 4). The NOE correlations of H 3 -15/H-6 and H-6/H-8 indicated they were cofacial and were arbitrarily assigned as α-orientations, whereas the correlations of H-7/H-9 and H-9/H 3 -14 showed their β-orientations. The NOE correlations of H-2/H-4 and H-6/H 3 -15, and the lack of correlation of H-4 with H-7, suggested that H-2, and 5-OH were β-oriented. Thus, the relative configuration of 1 was established.
Compound 2 possessed molecular formula of C 25 H 36 O 9 based on the HRESIMS ion at m/z 503.2261 [M + Na] + . The 1 H and 13 C NMR data of 2 were similar to those of 1, except that the isobutyryloxy groups at C-6 and C-9 in 1 were replaced by an angeloyloxy group at C-6 and a 3-methylbutyryloxy group at C-9 in 2. The 1 H-1 H COSY, HSQC, and HMBC spectra supported the structure of 2 as shown. The NOE correlations of H-2/H-4, H-6/H 3 -15, H-6/H-8, H-7/H-9 and H-9/H 3 -14 in 2 indicated that 2 had the same relative configuration as 1.
Compounds 3-4 shared the same molecular formula C 24 H 34 O 9 from their HRESIMS at m/z 489.2112 [M + Na] + and m/z 489.2100 [M + Na] + . The 1 H and 13 C NMR data of 3 showed a great similarity with those of 4, except for the ester residues at C-6. The angeloyloxy group at C-8 in 4 was placed by a tigloyloxy group in 3 24 . The 1 H-1 H COSY, HSQC and HMBC spectra of 3 confirmed this observation, leading to the assignment of its planar structure. The relative configuration of 3 was deduced to be the same as 4, on the basis of similar ROESY data.

Structural Elucidation of Compounds from Subtype II.
Herein, we further confirmed the absolute configurations of 10 and 11 by X-ray crystallographic analysis. Although a suitable single crystal of compound 10 or 11 was not obtained through many attempts with different solvents, mixed trigonal crystals of 10 and 11 (1:1) were obtained from MeOH. Both compounds have the same nuclear structures, and the only difference is that the absolute configuration of 2-methylbutyryloxy group at C-9 is 2″R or 2″S. Because the minor difference does not affect their crystal structures, the X-ray diffraction experiment of the mixture crystal supported the absolute configuration of the nuclear structure. The X-ray crystallographic analysis [flack parameter = −0.0(2)] unambiguously established the absolute configurations of 10 and 11 as 2S, 4R, 5R, 6R, 7S, 8S, 9R, and 10S (Fig. 6).
Considering In addition, compounds 10 and 11 were usually reported as a mixture from C. triste 25,26 . Although both of them were separated successfully in the literature 14 , the NMR data have not been reported. Herein, the NMR data of 10 and 11 were reported for the first time. The MS, 1 H NMR, and 13 CNMR spectroscopic data of compound 12 were consistent or superposable with those of divaricin B 8,19,20,26 . These data indicated that both of them shared the same relative configurations. However, all isolated compounds 1-11 and 13-14 had negative optical rotation, but divaricin B has the opposite optical rotation , which suggested that divaricin B and compound 12 could be enantiomers and have the opposite absolute configuration. Herein, in order to distinguish two compounds, compound 12 was named ent-divaricin B. The MS, NMR, CD and optical rotation data of 12 was also reported in the paper.
Similarly, there is confusion in the literature about this class of compounds (subtype II) due to the incorrect depiction of the epoxy bonds. Thus, the structures of germacranolides of this type (subtype II) were depicted correctly in this paper. However, the 1 H and 13 C NMR data implied that the structure of 15 was similar to that of 17 22,25 (supplementary information S24), except that two isobutyryloxy groups of 17 were replaced by two angeloyloxy groups in 15, which was further confirmed by the 1 H-1 H COSY, HSQC, and HMBC spectra (Fig. 3). The relative configuration of 15 was determined by analysis of ROESY data. The key NOE correlations of H-8/H-6, H-7/H-5, H-5/H 3 -15, H-7/H-9, and H-9/H-10 indicated that 15 had the same relative configuration as 17 (Fig. 4).

Structural Elucidation of Compounds from
Compound 16 was the isomers of 2, 7, 10, 11, 13, 18 and 19, based on its HRESIMS (m/z 503.2263 [M + Na] + , C 25 H 36 O 9 Na). The 1 H and 13 C NMR data were similar to those of 17, except for an angeloyloxy group at C-6 and the 3-methylbutyryloxy groups at c-9 in 16 instead of two isobutyryloxy groups in 17. The conclusion was confirmed by analysis of relevant 1 H-1 H COSY, HSQC and HMBC data. The relative configuration of 16 was determined to be the same as that of 15 by comparison of their ROESY data.
All compounds were evaluated for their cytotoxic activity against human cervical (HeLa), colon (LoVo), stomach (BGC-823), and breast cancer (MCF-7) cell lines.  1-3, 8-9, and 15-16), contained a 5-membered γ-lactone ring fused to a circular 10-membered carbocycle. Subtypes I and II have the same planar structure, but the absolute configurations at C-2 and C-5 are different (2R, 5S in subtype I and 2S, 5R in subtype II). We obtained six pairs of stereoisomers (1/8, 2/13, 4/14, 6/12, 7/11 and 10/11) from the same plant. The isolation of these stereoisomers is a huge challenge because they are highly oxygenated and have similar structures.        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.  difference peak/hole = 0.759/−0.445 e Å −3 , and flack parameter = −0.00 (11). The final R indexes [I > 2σ (I)] were R 1 = 0.0390, and wR 2 = 0.1006. The final R indexes (all data) were R 1 = 0.0397, and wR 2 = 0.1012. The goodness of fit on F 2 was 1.024. 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 μL of medium containing 0.1% DMSO or different concentrations of the test compounds and the positive control cis-platin. 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 29 . After addition of 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 3.