Two new eremophilane Sesquiterpenoides from Ligularia dictyoneura

Abstract Two new eremophilane sesquiterpenoides, 6α,9α-dihydroxyeremophilenolide (1), and 1β,10β-dihydroxyeremophilenolide (2), along with ten known eremophilane sesquiterpenoides (3–12) were isolated from the aerial parts of Ligularia dictyoneura (Franch.) Hand.-Mazz. Their structures were elucidated by means of extensive spectroscopic analysis. Compounds 3–6 were assessed for their cytotoxicity against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW-480), and the result showed that they had no activity. Graphical Abstract


Introduction
Ligularia is a genus of about 130 species in the family Compositae, mainly distributing in Asia, with 111 species being endemic to China (Liu 1989). The roots of several plants in the genus were usually used to treat syndromes of bronchitis, cough, tuberculosis and haemoptysis in traditional Chinese medicine. Ligularia plants are chemotaxonomically characteristic due to the presence of typical pyrrolizidine alkaloids and sesquiterpenoids. Among them, eremophilane-type sesquiterpenes are abundant and typical secondary metabolites found in this genus (Jia and Zhao 1994;Wu et al. 2005Wu et al. , 2016Liu et al. 2018), and some of the compounds exhibited significant cytotoxic and other activities. Ligularia dictyoneura (Franch.) Hand.-Mazz is distributed in the southwest of China. Previous Investigation on its constituents has isolated a lots of eremophilane sesquiterpenes (Zhao and Chen 1982;Tan et al. 2002;2003, 2004Nagano et al. 2007;Li et al. 2013). As a part of our research of structurally unique and bioactive compounds from medicinal plants of Yunnan, China, we have isolated and identified two new eremophilane sesquiterpenoides, 6a,9a-dihydroxyeremophilenolide (1), and 1b,10b-dihydroxyeremophilenolide (2), along with ten known eremophilane sesquiterpenoides (3-12) from the aerial parts of L. dictyoneura. Herein, we report the isolation and structural elucidation of compounds 1 and 2 ( Figure 1).

Results and discussion
Compound 1 was obtained as colourless amorphous powder. The molecular formula was determined as C 15 H 22 O 4 by the HR-ESI-MS at m/z 266.1523 ([M] þ ), indicating five degrees of unsaturation. Its IR spectrum revealed the presence of OH, C=O, and C=C groups from absorptions at 3427, 1739, and 1631 cm À1 , respectively. In the 1 H NMR spectrum, signals of three methyl groups were observed at d 0.83 (3H, d, J ¼ 6.5 Hz, H-15), 1.15 (3H, s, H-14) and 1.86 (3H, s, H-13). Its 13 C NMR and DEPT spectrum displayed 15 signals, including three methyls, three methylenes, five methines, and four quaternary carbons including one carbonyl and two olefinic carbons. These data suggested that 1 is an eremophilane sesquiterpene, similar to 10b-hydroxyeremophilenolide (3) (Massiot et al. 1990), which was also isolated in this study. The presence of three deshielded quaternary carbons at d 161.4 (C-7), 122.7 (C-11) and 176.9 (C-12) and of a vinylic methyl group suggested a methyl substituted a,b-unsaturated lactone with an endo-double bond. Other functional groups detected in these spectra were two secondary alcohol (d 73.8 and 69.6) and an oxygenated methine (d 86.1) involved in a lactone ring. Analysis of its 1 H-1 H COSY, HSQC, and HMBC spectra confirmed this assumption and helped furnish the planar structure of compound 1 ( Figure S1, Supplementary material). The HMBC correlations from H-6 (d 4.63) to C-4 (d 32.1), C-5 (d 44.9), C-7 (d 161.4), and C-8 (d 86.1), and from H-9 (d 3.66) to C-1 (d 21.1), C-8 (d 86.1) and C-10 (d 42.7) permitted the location of OH groups at C-6 and C-9, respectively. Due to biogenetic considerations of eremopilane derivatives isolated from Ligularia species, Me-14 and Me-15 were assigned to be b-orientation. ROESY correlations from H-14 to H-8 and H-10 revealed H-8 and H-10 also to be b-oriented. The b-orientation of H-6 was deduced from the presence of ROESY correlation of H-6 and Me-14. In the ROESY spectrum, H-9 correlated with H-8 and H-10 showing that one OH was located at C-9a. Thus, compound 1 was identified as 6a,9a-dihydroxyeremophilenolide.
Compound 2 was obtained also as colourless amorphous powder. The HR-ESI-MS exhibited a [M] þ peak at m/z 266.1524, which suggested a molecular formula of C 15 H 22 O 4 , indicating five degrees of unsaturation. The absorption bands in IR spectrum at 3527, 3493, 1728, and 1680 cm À1 accounted for the presence of OH, C=O, and C=C groups. Comparison of the NMR data of 2 with those of 3 indicated an additional secondary alcohol (d 75.1) was existed in 2. HMBC correlations from H-1 to C-2, C-3, C-5 and C-10 showed that a hydroxyl group attached to C-1. The relative configuration of H-1 was assigned as a-oriented by the ROSEY correlation between H-1 and H-9a. Thus, 2 was determined as 1b,10b-dihydroxyeremophilenolide.
Compounds 3-6 were assessed for their cytotoxicity against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW-480), but none of these isolates showed significant activity on the examined cancer cell lines. Compounds 1 and 2 were not evaluated for their cytotoxicity as their amounts were less.

Plant material
The aerial part of L. dictyoneura was collected from Sangri-la county of Yunnan Province in August, 2011. The identity of plant material was determined by Mr. Kang Min, a botanist of Diqing Institute of Agricultural Sciences, Sangri-la, where a voucher specimen (No. 1108012) was deposited.