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Drimane-type Sesquiterpenes with a Dioxabicyclooctane Skeleton from the Fruiting Bodies of Nigrofomes melanoporus and Their Cytotoxicity
Hsiu-Hui Chan 1*, Shin-Hun Juang 2, 3*, Tran Dinh Thang 4*, Min-Yu Chen 3, Ping-Chung Kuo 5, Mei-Lin Yang 6, Nguyen Thi Ngan 4, Nguyen Ngoc Linh 4, Tian-Shung Wu 1, 6, 7 1 Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung, Taiwan 2 Department of Medical Research, China Medical University Hospital, Taichung, Taiwan 3 Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan 4 Department of Chemistry, Vinh University, Vinh City, Nghean province, Vietnam 5 Department of Chemistry and Biotechnology, National Formosa University, Yunlin, Taiwan 6 Department of Chemistry, National Cheng Kung University, Tainan, Taiwan 7 Department of Pharmacy, China Medical University, Taichung, Taiwan

Fig. 1

Structures of nigrofomin A (1) and B (2).

Abstract
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Two new drimane-type compounds, nigrofomin A (1) and B (2), possessing a rare dioxabicyclooctane moiety, were purified from the fruiting bodies of Nigrofomes melanoporus. Their structures were determined using 1D, 2DNMR and HRESIMS spectroscopic analyses. In addition, 1 was established by Xray crystallographic studies. Both nigrofomin A (1) and B (2) exhibited cytotoxicity on acute T-cell leukemia (Jurkat), human nasopharyngeal carcinoma (NPC-TW01), and lung cancer (NCI-H661) cells with IC50 values in the range of 99.44246.32 M. Furthermore, the effects of 1 and 2 on cell-cycle progression of Jurkat cells displayed a concentration-dependent accumulation in the G0/G1 phase.

Key words Nigrofomes melanoporus Polyporaceae drimane sesquiterpenes cytotoxicity

Supporting information available online at http://www.thieme-connect.de/ejournals/toc/plantamedica

Polyporaceae, including Taiwanofungus, Ganoderma, and Poria genera, is a famous fungus family which belongs to the basidiomycete group. The constituents of the fruiting bodies of these fungi have been evidenced to be rich sources of biological properties, such as anti-inflammatory [1, 2], antihypertensive [3, 4], antitumor [59], and immunomodulatory and adjuvant activities [10]. We had previously reported the cytotoxicity of benzenoids and triterpenoids from Taiwanofungus camphorates [11, 12], triterpenoids from Ganoderma lucidum [13], and the chemical constituents from Cordyceps sinensis [14]. In our continuing program aimed to search for potential antitumor agents from natural sources, the

* These authors provided equal contributions to this work.

fruiting bodies of Nigrofomes melanoporus (Mont.) Murrill collected in Vietnam were subjected to chemical investigation and cytotoxicity studies. In the present paper, two new drimane-type sesquiterpenes with rarely appearing dioxabicyclooctane skeleton, nigrofomin A (1) and B (2), were identified with 1D, 2DNMR and HRESIMS spectroscopic analyses. To the best of our knowledge, this is the first report of the phytochemical constituents and biological activities of N. melanoporus. The air-dried fruiting bodies of N. melanoporus (1350 g) were extracted with the solvent mixture of chloroform and methanol at ambient temperature. The concentrated extract was subjected to silica gel column chromatography and further resolved with the aid of high-performance liquid chromatography to afford nigro" Fig. 1). fomin A (1) and B (2) (l Nigrofomin A (1) was obtained as optically active and colorless prisms, m.p. 208210 C. The HRESIMS of 1 displayed a quasi molecular ion peak at m/z 317.1002, which is consistent with a pseudomolecular formula of C15H18O6Na (calcd. 317.1001) and suggested seven degrees of unsaturation. The IR spectrum showed strong absorption peaks for hydroxyl (3360 cm1), a ketone carbonyl (1771 cm1), and a lactone carbonyl (1709 cm1). The 13CNMR, DEPT-135, and HSQC analyses of 1 displayed the presences of 15 carbon signals, including an aliphatic ketone carbon ( 210.9), a lactonic carbon ( 174.0), two olefinic carbons ( 126.3 and 136.3), four quaternary carbons ( 82.1, 76.7, 60.0, and 45.7), one methine ( 106.6), four methylenes ( 73.0, 52.5, 40.8, and 23.6), and two methyl ( 25.4 and 26.5) carbons. In the 1 HNMR spectrum of 1, two methyl singlets [ 1.31 (H-13, s), 1.15 (H-14, s)], an olefinic proton [ 6.02 (H-7, br s)], an oxygenbearing methylene [ 4.79 (1H, H-12, d, J = 10.4 Hz), 4.50 (1H, H12, d, J = 10.4 Hz)], three methylenes [ 3.53 (H-1, d, J = 13.2 Hz), 2.19 (H-1, d, J = 13.2 Hz), 3.08 (1H, H-3, d, J = 3.8 Hz), 1.91 (1H, H-3, d, J = 3.8 Hz), and 2.60 (2H, H-6, br s)], and an acetal signal [ 5.62 (H-11, s)] coupled with 13C signals at 25.4, 26.5, 126.3, 40.8, 52.5, 73.0, and 32.6, indicated the occurrence of a rare dioxabicyclooctane skeleton for a drimane-type sesquiterpene [15, 16]. The HMBC spectrum of 1 exhibited the cross-peaks of H-3 ( 3.10, 1.91) and C-2 ( 210.9)/C-4 ( 45.7)/ C5 ( 76.7)/C13 ( 25.4)/C14 ( 26.5), and H-13 ( 1.31)/H-14 ( 1.15) and C-3 ( 52.5)/C-4 ( 45.7)/C-5 ( 76.7). The 2J and 3J HMBC correlations indicated that the quaternary C-4 was substituted with two methyl groups. The downfield shifts of C-5 and C9 were due to the hydroxy substitutions. Furthermore, the dioxabicyclooctane moiety of 1 connected at C-8, 9, and 10 was established by the HMBC correlations of H-11 ( 5.62) with C-8 ( 136.3)/C12 ( 73.0)/C15 ( 174.0), H-12 ( 4.79, 4.50) with
Chan HH et al. Drimane-type Sesquiterpenes with Planta Med 2012; 78: 13

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PLAMED-2011-09-0867-LET (pmleV0867) Satz PDF 1 3. 3. 2012 Herst. eFirst n.a. Datum

Ziegler + Mller K. Grohe 03.03.2012

Fig. 2

Key NOE correlations of 1 and 2.

Fig. 3

Single-crystal Xray structure of 1.Better quality available?

C-7 ( 126.3)/C-8 ( 136.3)/C-9 ( 82.1), and H-1 ( 3.53, 2.19) with C-2 ( 210.9)/C-5 ( 76.7)/C-9 ( 82.1)/C10 ( 60.1)/C15 ( " Fig. 2) were observed be174.0). The key NOE correlations in 1 (l tween H-14 ( 1.15) and H-3 ( 1.91)/H-3 ( 3.08)/H-6 ( 2.60), H-13 ( 1.31) and H-3 ( 1.91), and H-6 ( 2.60) and H-7 ( 6.02). The relative configuration of 1 was further confirmed by an Xray " Fig. 3). Therefore, the stereodiffraction crystallographic study (l chemical structure of 1 was established as shown, and this compound has been named nigrofomin A (1). Nigrofomin B (2) was isolated as an optically active colorless powder and m.p. 213215 C. The IR absorption bands at 3368, 1771, and 1701 cm1 indicated the presence of hydroxyl, ketone, and lactone carbonyl groups, respectively. The 1HNMR spectrum of 2 was very similar to that of 1 except for the upfield shifts of H1 ( 2.27, d, J = 2.0 Hz), H-3 ( 2.20, dd, J = 12.8, 2.0 Hz), and H-13 ( 1.21, s) and presence of an H-5 signal at 3.19 (1H, d, J = 10.8 Hz). The HMBC correlations between H-5 ( 3.19) and C3 ( 56.7), C-9 ( 80.8), and C-15 ( 175.0) revealed that a hydroxy group was absent in C-5 of 2, supported by comparison of the molecular formulas of 1 (C15H18O6) and 2 (C15H18O5). In the NOESY spectrum of 2, the NOE correlations between H-11 ( 5.67) and H-3 ( 2.20)/H12 ( 4.79), H-5 ( 3.19) and H-3, H-3 ( 2.58) and H-14 ( 1.19), H-3 and H-13 ( 1.21), and H3 ( 2.20)/H6 ( 2.49) and H-13, constructed the relative " Fig. 2). It configuration of 2 which was the same as that of 1 (l
Chan HH et al. Drimane-type Sesquiterpenes with Planta Med 2012; 78: 13

" Fig. 1 or to is not clear if the preceding sentence should refer to l " Fig. 2 or both.Based on the above observations, the chemical l structure of nigrofomin B (2) was deduced as shown. To assess the growth inhibitory activity of nigrofomin A (1) (purity > 96 %) and B (2) (purity > 91%) towards tumor cell lines, three different cells from different organs, including acute T-cell leukemia (Jurkat), human nasopharyngeal carcinoma (NPCTW01), and lung cancer (NCI-H661) cell lines, were used. Treatment with compounds 1 and 2 showed inhibition of Jurkat cell lines with IC 50 values of 125 M and 99 M, respectively. However, NPC-TW01 and NCI-H661 cells were less susceptible to treatment with 1 and 2 with IC50 values ranging from 159 to 238 M for 1 and 188 to 246 M for 2, respectively. Furthermore, our results showed that only acute T-cell leukemia cells are sensitive to both 1 and 2 treatments with IC50 values around 100 M. Our results clearly demonstrated a significant increase of G0/G1 population with concomitant loss of the S phases when Jurkat cells are treated with both 1 and 2 for 48 hours. The accumulation of the G0/G1 population increased with treatment dosage enhancement, suggesting that the cell cycle arrest phenomena by 1 and 2 is in a concentration-dependent manner. This is the first report on the phytochemical study and biological activities of the Nigrofomes genus. The present study demonstrates that this genus fragment is a potential source of drimanetype sesquiterpenes possessing a rarely occurring dioxabicyclooctane fragment. Two new compounds, nigrofomin A (1) and B (2), showed slight cytotoxicity against the acute T-cell leukemia (Jurkat), NPC-TW01, and NCI-H661 cell lines, with IC50 values ranging from 99.44 to 246.32 M.

Materials and Methods

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The basidiomycete Nigrofomes melanoporus (Mont.) Murrill. was collected at the Pumat National Park of Nghean Province, Vietnam, in November 2009 and identified by Dr. Ngo Anh, Department of Biology, Hue University. A voucher specimen (Vin-TSWu 200 901) was deposited at the herbarium of the Department of Biology, Vinh University. The air-dried fruiting bodies (1350 g) were extracted three times with CHCl3/MeOH (1 : 1, v/v) at ambient temperature. After removal of the solvent by evaporation in vacuo, the brown residue (78.0 g) was subjected to silica gel column chromatography (1 kg, 80 8 cm), eluting with a hexaneacetone step gradient system

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(100 : 0, 50 : 1, 20 : 1, 10 : 1, 2 : 1, 1 : 1, each 1.0 L) to result in 10 fractions. Fraction 7 (5.4 g) was purified with the aid of silica gel column chromatography (200 g, 40 5 cm) eluting with a CHCl3 MeOH step gradient system (100 : 0, 50 : 1, 10 : 1, 2 : 1, 1 : 1, each 250 mL) to afford the sesquiterpene-rich subfraction. This subfraction (74 mg) was further resolved by RP-HPLC (H2O: MeOH = 65 : 35, v/v) using a 5-C18-MSII Waters column (20 250 mm) at a flow rate of 5.0 mL/min and detection at 197 nm to yield nigrofomin A (1) (13.2 mg) and B (2) (8.7 mg). The purities of compounds 1 and 2 were analyzed by reversed-phase HPLC (LichroCART 2544, Purospher STAR RP-8e, 5 m).

Supporting information
The experimental procedure and full characterization of compounds 1 and 2 are available online. The cytotoxicity assay is also available as Supporting Information.

11 Wu SJ, Leu YL, Chen CH, Chao CH, Shen DY, Chan HH, Lee EJ, Wu TS, Wang YH, Shen YC, Qian K, Bastow KF, Lee KH. Camphoratins AJ, potent cytotoxic and anti-inflammatory triterpenoids from the fruiting body of Taiwanofungus camphoratus. J Nat Prod 2010; 73: 17561762 12 Shi LS, Chao CH, Shen DY, Chan HH, Chen CH, Liao YR, Wu SJ, Leu YL, Shen YC, Kuo YH, Lee EJ, Qian K, Wu TS, Lee KH. Biologically active constituents from the fruiting body of Taiwanofungus camphoratus. Bioorg Med Chem 2011; 19: 677683 13 Wu TS, Shi LS, Kuo SC. Cytotoxicity of Ganoderma lucidum triterpenes. J Nat Prod 2001; 64: 11211122 14 Yang ML, Kuo PC, Hwang TL, Wu TS. Bioassay-guided purification of chemical constituents from the mycelia of Cordyceps sinensis and their bioactivities. J Nat Prod, in pressplease check, new info available? 15 Ayer WA, Craw PA, Stout TJ, Clardy J. Novel sesquiterpenoids from the fairy ring fungus, Marasmius oreades. Can J Chem 1989; 67: 773778 16 Ayer WA, Craw PA. Metabolites of the fairy ring fungus, Marasmius oreades. 2. Norsesquiterpenes, further sesquiterpenes, and agrocybin. Can J Chem 1989; 67: 13711380 received revised accepted September 9, 2011 February 10, 2012 February 17, 2012

Acknowledgements
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This study was supported by the National Science Council (Taiwan) and NAFOSTED (104.012010.27) (Vietnam), and in part by the Taiwan Development of Health Cancer Research Center of Excellence (DOH100-TD-B-111-004), DMR-96-082 (CMUH), and DMR-101105 (CMUH).

Bibliography DOI http://dx.doi.org/10.1055/s-0031-1298367 Planta Med 2012; 78: 13 Georg Thieme Verlag KG Stuttgart New York ISSN 00320943 Correspondence Prof. Tian-Shung Wu Department of Chemistry National Cheng Kung University 1 Ta-Hsueh Road Tainan 70101 Taiwan Phone: + 886 62 75 75 75 ext. 6 53 33 Fax: + 886 62 74 05 52 tswu@mail.ncku.edu.tw

Conflict of Interest
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The authors have no conflict of interest to report.

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Dear authors, please check the figures colour mode and supply 4-colour-data for figures, if necessary; the image data we received for the article (not the Supporting Information) were in black & white mode

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