Two new thymol derivatives from Eupatorium fortunei

Abstract Two new thymol derivatives (1–2) were isolated from the aerial parts of Eupatorium fortunei. Their structures were elucidated on the basis of comprehensive spectroscopic data analysis as 8,9-dehydrothymol-3-O-β-glucoside (1), and 9-(acetyloxy)thymol-3-yl (3-methylbut-2-enoate) (2). All isolates were evaluated for cytotoxic activities with IC50 values greater than 50 μM in vitro against MCF-7, HeLa, A549, and Hep G-2 cancer cells. Graphical Abstract


Introduction
Eupatorium fortunei Turcz., belonging to the family Eupatorium, is a perennial herb, widely distributed in China.The stem and leaf of E. fortunei are commonly used as folk herbal medicine for treating dropsy, fever, chills and nausea (Chinese Pharmacopoeia Commission 2015).Previous phytochemical investigations have afforded many compounds with various structural patterns, such as essential oil (Zhu et al. 2008;Li et al. 2020), terpenoids (Jiang and Gao 2005;Jiang et al. 2006;Chen et al. 2013;Lee et al. 2020;Shi et al. 2020), thymol derivatives (Tori et al. 2001;Wang et al. 2014;Pham et al. 2019;Yu et al. 2020), alkaloids (Liu et al. 1992;Zhang et al. 2021), and benzofuran (Jiang et al. 2008;Chang et al. 2021).Some compounds and the extract of E. fortunei displayed potential biological activities, including cytotoxic (Lee et al. 2020;Yu et al. 2020;Chang et al. 2021;Zhang et al. 2021), anti-bacterial (Pham et al. 2019;Li et al. 2020), anti-inflammatory (Wang et al. 2014;Shi et al. 2020), antiviral (Choi et al. 2017), and anti-tumor (Kim et al. 2014) activities.As part of our investigation to find diverse and bioactive compounds from E. fortunei, our previous investigations led to the isolation of four new thymol derivatives, one new isothymol derivative with cytotoxicities (Yu et al. 2020), and two pairs of unusual monoterpene enantiomers with anti-inflammatory activities (Shi et al. 2020).Our continued phytochemical studies of E. fortunei afforded two additional new thymol derivatives.Herein, we report the isolation, structural elucidation of these compounds, and the evaluation of their cytotoxic activities on MCF-7, HeLa, A549, and Hep G-2 cells.
Although compound 2 has a chiral center at the 8-position, no optical rotation was observed.Analysis using a chiral HPLC column compound 2 showed two peaks (Supplementary material, Figure S14), which revealed compound 2 exists as racemic mixtures.
Compounds 1-2 were evaluated for their cytotoxic activities against MCF-7, HeLa, A549, and Hep G-2.Unfortunately, the results suggested that compounds 1-2 displayed no obvious cytotoxicity with IC 50 values greater than 50 lM against human cancer cell lines.

Plant material
The aerial parts of E. fortunei were purchased from Kangmei Pharmaceutical Co. Ltd in Bozhou, Anhui Province.A voucher specimen (No. 20170912) was preserved in the Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences.

Acid hydrolysis of compounds 1
Compound 1 (2.0 mg) was refluxed with 2.0 mL 2.0 M HCl-H 2 O at 80 C for 5 h and then extracted with EtOAc (1.0 mL Â 3).The H 2 O layer was evaporated in vacuo to furnish a neutral residue.The residue was dissolved in pyridine (1.0 mL), and then added 2.0 mg of L-cysteine methyl ester hydrochloride.The mixture was stirred at 60 C for 2 h, and after evaporation in vacuo to dryness, 0.2 mL of N-trimethylsilylimidazole was added; the mixture was refluxed at 60 C for another 2 h.The reaction mixture was partitioned between n-hexane and H 2 O (2.0 mL each), and the n-hexane extract was analyzed by GC under the following conditions: capillary column, HP-5 (30 m Â 0.25 mm, with a 0.25 lm film, Dikma); detection, FID; detector temperature, 280 C; injection temperature, 250 C; initial temperature 160 C, then raised to 280 C at 5 C/min, final temperature maintained for 10 min and carrier, N 2 gas.The standard D-glucose was treated by the same reaction and gas chromatographic conditions.As a result, D-glucose (t R 19.1 min) of 1 was detected by comparing their retention times with the authentic samples D-glucose (t R 19.1 min) (Shi et al. 2012).

Cytotoxicity assay
Cytotoxic activities of compounds 1-2 against MCF-7, HeLa, A549, and Hep G-2, were evaluated using MTT assay.The anticancer agent cisplatin was used as a positive control.Briefly, cancer cells were first seeded into 96-well plates at 3000 cells/well, 100 lL/ well and incubated for 24 h.Next, the cells were treated with compounds 1-2 for 48 h.Then, MTT solution (5.0 mg/mL) was added into each well and the cells were incubated for another 2 h.Finally, DMSO was added and the optical density was measured at 570 nm using an ELx800 microplate reader (Bio-Tek, Winooski, VT, USA) with subtraction of background absorbance.Compounds 1-2 were tested at five concentrations and were dissolved in DMSO to give a final DMSO concentration of 0.1% in each well.Each concentration of the compounds was tested in three parallel wells and took the average.The 50% inhibitory concentration (IC 50 ) values were calculated (Yu et al. 2020).

Conclusions
In summary, two new thymol derivatives (1-2) were isolated from the aerial parts of E. fortunei.Their structures were determined by comprehensive spectroscopic analysis.Unfortunately, all isolates displayed no obvious cytotoxicity with IC 50 values greater than 50 lM against human cancer cell lines.Combined with our previous studies (Yu et al. 2020), it seems that various thymol derivatives are the major chemical constituents of E. fortunei and minor changes in the structure affect their activities.The absences of the hydroxyl group at C-3 position of compounds 1, 2 decrease their cytotoxic activities.Based on these findings, the search for active compounds could be guided in the future.