International Journal of Molecular Sciences Article Dibenzofuran, 4-Chromanone, Acetophenone, and Dithiecine Derivatives: Cytotoxic Constituents from Eupatorium fortunei Chun-Hao Chang 1, Semon Wu 2, Kai-Cheng Hsu 3,4, Wei-Jan Huang 5,6 and Jih-Jung Chen 7,8,9,* 1 Institute of Biopharmaceutical Sciences, School of Pharmaceutical Sciences, National Yang Ming Chiao Tung University, Taipei 112, Taiwan; [email protected] 2 Department of Life Science, Chinese Culture University, Taipei 110, Taiwan; [email protected] 3 Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan; [email protected] 4 Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan 5 Ph.D. Program in Biotechnology Research and Development, College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan; [email protected] 6 Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan 7 Department of Pharmacy, School of Pharmaceutical Sciences, National Yang Ming Chiao Tung University, Taipei 112, Taiwan 8 Faculty of Pharmacy, National Yang-Ming University, Taipei 112, Taiwan 9 Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan * Correspondence: [email protected]; Tel.: +886-2-2826-7195; Fax: +886-2-2823-2940 Abstract: Five new compounds, eupatodibenzofuran A (1), eupatodibenzofuran B (2), 6-acetyl-8- methoxy-2,2-dimethylchroman-4-one (3), eupatofortunone (4), and eupatodithiecine (5), have been isolated from the aerial part of Eupatorium fortunei, together with 11 known compounds (6-16). Citation: Chang, C.-H.; Wu, S.; Hsu, K.-C.; Huang, W.-J.; Chen, J.-J. Compounds 1 and 2 featured a new carbon skeleton with an unprecedented 1-(9-(4-methylphenyl)-6- Dibenzofuran, 4-Chromanone, methyldibe nzo[b,d]furan-2-yl)ethenone. Among the isolates, compound 1 exhibited potent inhibitory Acetophenone, and Dithiecine activity with IC50 values of 5.95 ± 0.89 and 5.55 ± 0.23 µM, respectively, against A549 and MCF-7 Derivatives: Cytotoxic Constituents cells. The colony-formation assay demonstrated that compound 1 (5 µM) obviously decreased A549 from Eupatorium fortunei. Int. J. Mol. and MCF-7 cell proliferation, and Western blot test confirmed that compound 1 markedly induced Sci. 2021, 22, 7448. https://doi.org/ apoptosis of A549 and MCF-7 cells through mitochondrial- and caspase-3-dependent pathways. 10.3390/ijms22147448 Keywords: Eupatorium fortunei; dibenzofuran; cytotoxity; apoptosis Academic Editor: Toshio Morikawa Received: 26 May 2021 Accepted: 7 July 2021 1. Introduction Published: 12 July 2021 Eupatorium fortunei Turcz. (Asteraceae) is a perennial herb widely distributed in China. E. fortunei Publisher’s Note: MDPI stays neutral The aerial part of (Chinese name: Pei-Lan) has been used as a traditional medicine with regard to jurisdictional claims in for the treatment of various diseases such as flu, poor appetite, constipation, nausea, and published maps and institutional affil- siriasis [1]. Diverse monoterpenoids [2–6], triterpenoids [2,7], sesquiterpenoids, stereoiso- iations. mer, coumarins [7], pyrrolizidine alkaloids [7,8], benzofuran [4,9], and their derivatives were isolated from this species in the past studies. Many of these isolated compounds showed anti-inflammatory [5], anti-bacterial [3,4], anti-cancer [6], and anti-diabetic [7] activities. In our study on the cytotoxic constituents of Chinese herbal medicines, many species have been screened for cytotoxic effect, and E. fortunei has been found to be an Copyright: © 2021 by the authors. b,d 1 Licensee MDPI, Basel, Switzerland. active species. Two new dibenzo[ ]furan derivatives, eupatodibenzofuran A ( ) and This article is an open access article eupatodibenzofuran B (2), a new 4-chromanone, 6-acetyl-8-methoxy-2,2-dimethylchroman- distributed under the terms and 4-one (3), a new acetophenone derivative, eupatofortunone (4), a new dithiecine derivative, conditions of the Creative Commons eupatodithiecine (5), and eleven known compounds (6–16) have been isolated and con- Attribution (CC BY) license (https:// firmed from the aerial part of E. fortunei. This report describes the structural elucidation creativecommons.org/licenses/by/ of five new compounds 1–5 and the inhibitory activities of 1 against non-small-cell lung 4.0/). cancer and breast cancer cells. Int. J. Mol. Sci. 2021, 22, 7448. https://doi.org/10.3390/ijms22147448 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 17 Int. J. Mol. Sci. 2021, 22, 7448 This report describes the structural elucidation of five new compounds 1–5 and2 of 16the in- hibitory activities of 1 against non-small-cell lung cancer and breast cancer cells. 2. Results and Discussion 2. Results and Discussion 2.1.2.1. General General SeparationSeparation of of the the EtOAc-soluble EtOAc-soluble fraction fraction of aof MeOH a MeOH extract extract of the of aerialthe aerial part ofpartE. of E. fortuneifortuneiby by silica silica gel gel chromatography chromatography and and preparative preparative thin-layer thin-layer chromatography chromatography (TLC) (TLC) affordedafforded five five new new (1 –(51)–5 and) and eleven eleven known known compounds compounds (6–16 (6)– (Figure16) (Figure1). 1). FigureFigure 1. 1.The The chemical chemical structures structures of new of new compounds compounds1–5 and 1–5known and known compounds compounds6–16 isolated 6–16 isolated from E.from fortunei E. fortunei. Int. J. Mol. Sci. 2021, 22, 7448 3 of 16 2.2. Structure Elucidation of the New Compounds Compound 1 was obtained as colorless needles. Its molecular formula C22H18O4 was deduced from a sodium adduct ion at m/z 369.11041 [M + Na]+ (calcd 369.11028) in the HRESIMS spectrum. The presence of hydroxyl (3439 cm−1) and conjugated carbonyl (1645 cm−1) groups were revealed from the IR spectrum. Analysis of the 1H and 13C NMR data of 1 showed the signals for an acetyl group [δH 2.41 (3H, s, Ac-2); δC 26.2 (COMe), 203.9 (COMe)], a chelated hydroxyl group [δH 12.65 (1H, s, OH-3)], a methyl group [δH 2.62 (3H, s, Me-6); δC 15.1 (Me-6)], a 2-hydroxy-4-methylphenyl group [δH 2.44 (3H, s, Me-40), 4.97 (1H, s, OH-20), 6.93 (1H, br d, J = 7.5 Hz, H-50), 6.96 (1H, br s, 0 0 0 0 0 0 H-3 ), 7.25 (1H, d, J = 7.5 Hz, H-6 ); δC 21.4 (Me-4 ), 116.1 (C-3 ), 121.7 (C-5 ), 122.4 (C-1 ), 0 0 0 130.2 (C-6 ), 140.4 (C-4 ), 152.7 (C-2 )], two ortho-coupled aromatic protons [δH 7.22 (1H, d, J = 7.5 Hz, H-8), 7.33 (1H, br d, J = 7.5 Hz, H-7); δC 125.1 (C-8), 128.6 (C-7)], and two singlet aromatic protons [δH 7.07 (1H, s, H-4), 7.65 (1H, s, H-1); δC 99.8 (C-4), 125.4 (C-1)] (Table1). The position of each substituent was supported by ROESY correlations between 0 0 H-1 (δH 7.65)/Ac-2 (δH 2.41), Me-6 (δH 2.62)/H-7 (δH 7.33), OH-2 (δH 4.97)/H-3 (δH 0 0 0 0 6.96), H-3 (δH 6.96)/Me-4 (δH 2.44), and Me-4 (δH 2.44)/H-5 (δH 6.93) and by HMBC correlation between ‘Ac-2 (δH 2.41)/C-2 (δC 116.6)’, ‘OH-3 (δH 12.65)/C-2 (δC 116.6), C-4 0 0 (δC 99.8)’, ‘Me-6 (δH 2.62)/C-5a (δC 155.9), C-7 (δC 128.6)’, ‘OH-2 (δH 4.97)/C-3 (δC 116.1)’, 0 0 0 and ‘Me-4 (δH 2.44)/C-4 (δC 140.4), C-5 (δC 121.7)’ (Figure2). The full assignment of 1H and 13C NMR resonances was supported by 1H–1H COSY, HSQC, ROESY (Figure2A ), and HMBC (Figure2B) spectral analyses. Thus, the structure of 1 was elucidated as 1-(3- hydroxy-9-(2-hydroxy-4-methylphenyl)-6-methyldibenzo[b,d]furan-2-yl)ethenone, named eupatodibenzofuran A. Table 1. 1H and 13C NMR data of 1 and 2. 1 2 a b a b Position δH (J in Hz) δC δH (J in Hz) δC 1 7.65, s 125.4 7.59, s 125.1 2 116.6 116.0 3 163.2 162.9 4 7.07, s 99.8 7.05, s 99.6 4a 161.2 161.3 5a 155.9 155.4 6 121.8 120.5 7 7.33, br d (7.5) 128.6 7.27, br d (7.6) 127.9 8 7.22, d (7.5) 125.1 7.17, d (7.6) 125.0 9 127.7 130.6 9a 121.9 121.7 9b 116.3 117.8 10 122.4 125.4 20 152.7 156.7 30 6.96, br s 116.1 6.91, br s 111.5 40 140.4 139.8 50 6.93, br d (7.5) 121.7 6.95, br d (7.5) 121.4 60 7.25, d (7.5) 130.2 7.28, d (7.5) 131.0 COMe-2 203.9 203.7 COMe-2 2.41, s 26.2 2.40, s 26.2 OH-3 12.65, s 12.62, s Me-6 2.62, s 15.1 2.60, s 15.1 OH-20 4.97, s OMe-20 3.70, s 55.5 Me-40 2.44, s 21.4 2.49, s 21.7 a b Recorded in CDCl3 at 500 MHz.