Natural Product Research, 2018 https://doi.org/10.1080/14786419.2018.1434641
Daedaleanols A and B, two new sesquiterpenes from cultures of the basidiomycete Daedalea incana Ying Huanga,b,c, Shuai-Bing Zhanga, He-Ping Chena, Zhen-Zhu Zhaob,c, Zheng-Hui Lia, Tao Fenga and Ji-Kai Liua a
School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan, China; bState Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China; cUniversity of Chinese Academy of Sciences, Beijing, China
ABSTRACT
Two new sesquiterpenes, daedaleanols A (1) and B (2), together with three known sesquiterpenes (3–5), were isolated from cultures of the basidiomycete Daedalea incana. Their structures were elucidated on the basis of extensive spectroscopic means. All compounds were tested for their cytotoxicities against three human cancer cell lines.
ARTICLE HISTORY
Received 6 December 2017 Accepted 21 January 2018 KEYWORDS
Daedalea incana; sesquiterpenes; cytotoxicity
1. Introduction The genus Daedalea, belonging to the order Polyporales and family Fomitopsidaceae, is widely distributed throughout the world. Previous chemical investigations on the genus Daedalea mainly reported chromenes, like quercinol (Gebhardt et al. 2007) and daedalin A (Morimura et al. 2009), and a series of triterpenoids (Kawagishi et al. 1997; Rosecke and Konig 2000; Yoshikawa et al. 2005), such as daedaleanic acids A–E (Ríos et al. 2012), daedaleasides (Ríos et al. 2012) and daedalols A–C (Sorribas et al. 2011). They exhibit diverse bioactivities, including anti-inflammatory (Gebhardt et al. 2007), antifungal (Ilondu 2013), antioxidant CONTACT Tao Feng
[email protected]; Ji-Kai Liu
[email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1434641. © 2018 Informa UK Limited, trading as Taylor & Francis Group
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(Sharma et al. 2010; Lee et al. 2013; Mao et al. 2014), anti-CML (Khamaisie et al. 2011), cytotoxic (Ríos et al. 2012), melanogenesis and tyrosinase inhibitory activity (Morimura et al. 2009). Daedalea incana is widespread in Asia which exhibits a brown rot in the attacked wood (Dai 2012). However, to the best of our knowledge, there was very little study, and simultaneously, no chemical investigation has been reported about this species. In this paper, we report the isolation and structure elucidation of two new sesquiterpenes, daedaleanols A (1) and B (2), together with three known sesquiterpenes (3–5) from the cultures of the basidiomycete D. incana (Figure 1). All of these compounds were evaluated for their cytotoxicities against three human cancer cell lines.
2. Results and discussion Compound 1 was obtained as a colourless oil, in which the molecular formula was established as C15H28O2 by HRESIMS (m/z 263.1982 [M + Na]+, Calcd 263.1982), requiring two degrees of unsaturation. The IR spectrum showed the presence of both olefinic groups (1632 cm−1) and hydroxy groups (3411 cm−1). Three doublets of doublets at δ 5.91 (1H), 5.19 (1H), and 5.02 (1H) showed a coupling pattern characteristic of a terminal vinyl group (Inui et al. 2010) possessing a trans vicinal coupling (J = 17.4 Hz), a cis vicinal coupling (J = 10.8 Hz), and a small geminal coupling (J = 1.4 Hz). The 1H NMR spectrum of 1 also indicated the presence one oxygenated methylene (δH 3.41, 3.31) and three methyl groups (δH 1.59, 1.25, 0.90). The 13C NMR spectrum showed the occurrence of four olefinic carbons (δC 146.3, 136.1, 125.7, 112.0). According to the degree of unsaturation, it was simply identified that 1 was a chain structure. Besides, 1 possessed one oxygenated quaternary carbon (δC 73.9) and one oxygenated methylene carbon (δC 68.5). Analysis of extensive 2D NMR data led to the
Figure 1. Structures of compounds 1–5.
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structural assignment of 1. The 1H–1H COSY cross-peaks were observed for H-1/H-2, H-4/H-6, H-6/H-7, H-9/H-11, H-11/H-12, H-12/H-13, H-13/H-14 and H-13/H-15, combined with the HMBC cross-peaks from H-5 (δH 1.25) to C-2 (δC 146.3) and C-4 (δC 43.5), form H-10 (δH 1.59) to C-7 (δC 125.7) and C-9 (δC 41.0), from H-15 (δH 0.90) to C-12 (δC 33.9) and C-14 (δC 68.5) confirmed the chain structure between C-1 and C-14. Therefore, the structure of 1 was determined as (E)-2,6,10-trimethyldodeca-6,11-diene-1,10-diol and named daedaleanol A. Compound 2 was isolated as colourless oil. Its molecular formula C20H31NO3 was informed from HRESIMS data (m/z 356.2194 [M + Na]+, Calcd 356.2196). The IR spectrum indicated the presence of both carbonyl (1736 cm−1) and olefinic groups (1632 cm−1). Compound 2 had a vinyl proton (δH 4.86, s; 4.53, s; δC 107.6, t) and three methyl groups at higher fields (δH 0.90, s; 0.85, s; 0.81, s). The correlation of H-1/H-2/H-3, and H-5/H-6/H-7, H-9/H-11 in the 1H–1H COSY spectrum, together with the correlation from H-12 to C-3 and C-5, and from H-15 to C-7 and C-9, as well as from H-14 to C-1, C-9 and C-5 in the HMBC spectrum were observed, which suggested that compound 2 owned a drimane-sesquiterpenoid skeleton. Additionally, in the 1H–1H COSY spectrum, the correlation from H-3′ to H-2′ and H-4′, and the correlation between H-3′ and C-1′, C-5′ in the HMBC spectrum, revealed that a fragment structure of pyroglutamate exited. Further analysis of the NMR data for the C5 moiety led to the conclusion that it was 2(S)-pyroglutamate, which was in good agreement with 1H and 13C NMR data in the literature (Parrish and Mathias 2002; Zhou et al. 2008). Similarly, NMR data for C15 moiety suggested it was albicanol (Toyota et al. 1994; Hoshino et al. 2004). Compound 2 was the condensation product of these two moieties, since the key difference between 2 and albicanol was that the C-11 (δC 63.5) occurred lowfield and there was a correlation from H-11 (δH 4.43, 4.32) to an ester carbonyl carbon C-1′ (δC 174.1). In the ROESY spectrum, the correlations between H-5 (δH 1.20) and H-9 (δH 2.12), H-12 (δH 0.90), suggested an α-orientation of H-9. Besides, there were obvious ROESY correlations between H-14 (δH 0.81) and H-11 (δH 4.43, 4.32), H-13 (δH 0.85). Thus, the relative configuration of albicanol fragment in 2 was determined, which proposed to be the same as that in the literature (Toyota et al. 1994; Hoshino et al. 2004). Therefore, compound 2 was identified and named daedaleanol B. The known compounds, albicanol (3) (Toyota et al. 1994; Hoshino et al. 2004), isodrimenediol (4) (Fleck et al. 1996), and neroplomacrol (5) (Inui et al. 2010), were identified by comparison of their spectroscopic data with those reported. All compounds were evaluated for their cytotoxicities against three human cancer cell lines, however, none of them showed activity (>40 μM).
3. Experimental section 3.1. General experimental procedures NMR, Bruker AV-400 instrument and Bruker Avance III 600 MHz spectrometer (Bruker Biospin GmbH, Karlsruhe, Germany); HRESIMS, Agilent 6200 Q-TOF MS system (Agilent Technologies, Santa Clara, CA, USA); OR, Jasco-P-1020 polarimeter (Horiba, Kyoto, Japan); UV, Shimadzu UV-2401PC (Shimadzu, Kyoto, Japan); IR, Bruker Tensor 27 FT-IR spectrometer; CC, Sephadex LH-20 (Amersham Biosciences, Upssala, Sweden) and silica gel (Qingdao Haiyang Chemical Co., Ltd); MPLC, Büchi Sepacore System equipping with pump manager C-615, pump modules C-605 and fraction collector C-660 (Büchi Labortechnik AG, Flawil, Switzerland), and columns packed with Chromatorex C-18 (40–75 μm, Fuji Silysia Chemical Ltd., Kasugai,
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Japan); Prep-HPLC, Agilent 1260 liquid chromatography system equipped with Zorbax SB-C18 columns (5 μm, 9.4 mm × 150 mm or 21.2 mm × 150 mm) and a DAD detector.
3.2. Fungal material The fungus D. incana was collected from Changjiang County, Hainan Province, China, in September 2012, and identified by Zhenghui Li, South-Central University for Nationalities (SCUN). The voucher specimen (no. Dai12039) was deposited at the School of Pharmaceutical Sciences, SCUN. Culture medium: peptone (0.15%), glucose (5%), KH2PO4 (0.05%), MgSO4 (0.05%), yeast powder (0.5%). Fermentation was carried out on a shaker at 25 °C and 150 rpm for 25 days.
3.3. Extraction and isolation The culture broth (20 L) was filtered, and the filtrate was condensed in vacuo and extracted with EtOAc (3 × 5 L), while the mycelium was extracted with CHCl3-MeOH (1:1, 3 × 2 L). The EtOAc extracts were evaporated under reduced pressure to give a crude extract (7.2 g), which was subjected to MPLC, eluted with a stepwise gradient of MeOH/H2O (v/v 20:80-100:0) to afford to afford fractions A-E. Fraction E was further separated by Sephadex LH-20 (MeOH) column chromatography to afford seven subfractions (E1-E7). Compound 1 (4.2 mg) was obtained from fraction E5 by prep-HPLC using a gradient eluent (MeCN–H2O 35%–50%, 25 min). Compounds 2 (2.1 mg) and 3 (8.5 mg) were yield from fraction E2 and E4 respectively by prep-HPLC using a gradient eluent (MeCN–H2O 65%–80%, 20 min). Fraction E3 was subjected on prep-HPLC (MeCN–H2O 25%–40%, 25 min) to afford Compound 4 (9.1 mg). E6 was purified by using prep-HPLC (MeCN-H2O 20%-35%, 25 min) to give compound 5 (4.5 mg).
3.4. Daedaleanol A (1) Colourless oil, [𝛼]20 D − 12.1(c 0.11, MeOH). UV (MeOH) λmax nm (log ε): 203 (3.46). IR (KBr) νmax −1 cm : 3411, 2930, 2873, 1632, 1384, 1042. HRESIMS m/z 263.1982 [M + Na]+ (Calcd for C15H28NaO2, 263.1982). 1H NMR data (400 MHz, methanol-d4): δH 5.19 (1H, dd, J = 17.4, 1.4 Hz, H-1), 5.02 (1H, dd, J = 10.8, 1.4 Hz, H-1), 5.91 (1H, dd, J = 17.4, 10.8 Hz, H-2), 1.51 (2H, m, H-4), 1.25 (3H, s, H-5), 2.02 (2H, m, H-6), 5.12 (1H, t, J = 6.7 Hz, H-7), 1.96 (2H, m, H-9), 1.59 (3H, s, H-10), 1.48 (1H, m, H-11), 1.39 (1H, m, H-11), 1.36 (1H, m, H-12), 1.04 (1H, m, H-12), 1.55 (1H, m, H-13), 3.41 (1H, dd, J = 10.6, 5.8 Hz, H-14), 3.31 (1H, m, H-14), 0.90 (3H, d, J = 6.7 Hz, H-15). 13 C NMR data (100 MHz, methanol-d4): δC 112.0 (C-1, t), 146.3 (C-2, d), 73.9 (C-3, s), 43.5 (C-4, t), 27.6 (C-5, q), 23.7 (C-6, t), 125.7 (C-7, d), 136.1 (C-8, s), 41.0 (C-9, t), 15.9 (C-10, q), 26.4 (C-11, t), 33.9 (C-12, t), 36.8 (C-13, d), 68.5 (C-14, t), 17.1 (C-15, q).
3.5. Daedaleanol B (2) Colourless oil, [𝛼]20 D + 6.2 (c 0. 18, MeOH). UV (MeOH) λmax nm (log ε): 202 (3.58). IR (KBr) νmax cm−1: 3446, 2927, 1736, 1632, 1384, 1046. HRESIMS m/z 356.2194 [M + Na]+ (Calcd for C20H31NNaO3, 356.2196). 1H NMR data (600 MHz, methanol-d4): δH 1.73 (1H, m, H-1), 1.26 (1H, m, H-1), 1.61 (1H, m, H-2), 1.52 (1H, m, H-2), 1.41 (1H, m, H-3), 1.24 (1H, m, H-3), 1.20 (1H, dd,
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J = 12.6, 2.6 Hz, H-5), 1.75 (1H, m, H-6), 1.35 (1H, m, H-6), 2.42 (1H, m, H-7), 2.05 (1H, m, H-7), 2.12 (1H, m, H-9), 4.43 (1H, dd, J = 11.1, 3.9 Hz, H-11), 4.32 (1H, dd, J = 11.1, 9.1 Hz, H-11), 0.90 (3H, s, H-12), 0.85 (3H, s, H-13), 0.81 (3H, s, H-14), 4.86 (1H, s, H-15), 4.53 (1H, s, H-15), 4.23 (1H, dd, J = 9.1, 4.1 Hz, H-2′), 2.42 (1H, m, H-3′), 2.13 (1H, m, H-3′), 2.32 (1H, m, H-4′), 2.28 (1H, m, H-4′). 13C NMR data (150 MHz, methanol-d4): δC 40.2 (C-1, t), 20.2 (C-2, t), 43.1 (C-3, t), 34.4 (C-4, s), 56.3 (C-5, d), 25.1 (C-6, t), 38.7 (C-7, t), 148.2 (C-8, s), 56.2 (C-9, d), 40.1 (C-10, s), 63.5 (C-11, t), 34.1 (C-12, q), 22.2 (C-13, q), 15.6 (C-14, q), 107.6 (C-15, t), 174.1 (C-1′, s), 57.2 (C-2′, d), 25.9 (C-3′, t), 30.2 (C-4′, t), 180.9 (C-5′, s).
3.6. Cytotoxicity assay The cytotoxicities against human myeloid leukaemia HL-60, hepatocellular carcinoma SMMC7721, lung cancer A-549 cells lines of compounds 1 to 5 were tested by using the MTS (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium) method (Mosmann 1983). MTS was an analogue of MTT, which could be reduced into soluble formazan by succinate dehydrogenase in mitochondria of living cells. Moreover, the optical density value of formazan (490 nm) was proportional to the number of living cells.
4. Conclusion Five sesquiterpenes, including two new ones, daedaleanols A and B, were isolated from cultures of the basidiomycete D. incana for the first time. All compounds were evaluated for their cytotoxicities against three human cancer cell lines. Even though none of compounds showed activity, this study improved phytochemical knowledge about the basidiomycete D. incana.
Supplementary material Table of 13C NMR for compounds 1–5; NMR spectra and HRESIMS of compounds 1 and 2.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work is supported by the National Natural Science Foundation of China [grant number 81561148013], [grant number 81373289].
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