Miscellaneous terpenoid constituents of Abiesnephrolepis and their moderate cytotoxic activities

Phytochemistry 72 (2011) 2197–2204

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Miscellaneous terpenoid constituents of Abies nephrolepis and their moderate cytotoxic activities Dan-Wei Ou-Yang a,b, Liang Wu a, Yong-Li Li d, Pei-Ming Yang b, De-Yun Kong b, Xian-Wen Yang c,⇑, Wei-Dong Zhang a,d,⇑ a

Department of Natural Product Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China Department of Chinese Traditional Medicine, Shanghai Institute of Pharmaceutical Industry, 1320 West Beijing Road, Shanghai 200040, China Key Laboratory of Marine Bio-resources Sustainable Utilization, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbes, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China d School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China b c

a r t i c l e

i n f o

Article history: Received 24 December 2010 Received in revised form 24 July 2011 Available online 27 August 2011 Keywords: Abies nephrolepis (Trautv.) Maxim Pinaceae Terpenoid Cu-Ka X-ray crystallography Cytotoxicity

a b s t r a c t Three monoterpenoids and two triterpenoids were isolated from Abies nephrolepis together with 53 known terpenoids. The structures of the compounds were established by 1D and 2D NMR spectroscopy. The absolute configuration of 3-hydroxycamphane-2-carboxylic acid was established as (1S,2R,3S,4R) by Cu-Ka X-ray crystallography. All 58 isolates were tested for cytotoxic activity against four tumor cells viz. A549 (human lung adenocarcinoma), Colo205 (colon adenocarcinoma), QGY-7703 (human hepatoma) and THP-1 (human monocytic leukemia). a-Cadinol exhibited the best effects on A549, Colo205 and QGY-7703 with IC50 values of 8.6, 8.1 and 4.6 lg/mL, respectively. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Abies is an important genus of the Pinaceae family. It is distributed widely in the highlands of Asia, Europe, North and Middle America, and North Africa. Some species were reported to possess a broad spectrum of antitumor, antibacterial, and anti-inflammatory bioactivities (Yang et al., 2008b). Since 2006, our research interests have focused on this species, and have led to the isolation of a variety of bioactive compounds with novel structures (Li et al., 2009a,b; Yang et al., 2009, 2008a, 2010b). In continuation of these studies, Abies nephrolepis (Trautv.) Maxim, a plant largely distributed in Xiaoxinganling and Changbai Mountains of the Northeastern China (Zheng and Fu, 1978), was selected for phytochemical and biological investigations. As a result, 58 compounds were discovered, including 13 monoterpenoids, four apocarotenoids, 9 sesquiterpenoids, one norditerpenoid, 15 diterpenoids, and 16 triterpenoids. In this paper, the isolation and structural elucidation ⇑ Corresponding authors at: Key Laboratory of Marine Bio-resources Sustainable Utilization, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbes, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China. Tel.: +86 (0) 20 89023 3174; fax: +86 (0) 20 8445 1672 (X.-W. Yang), Department of Natural Product Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China. Tel./fax: +86 (0) 21 8187 1244 (W.-D. Zang). E-mail addresses: [email protected] (X.-W. Yang), [email protected] (W.-D. Zhang). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.08.003

of five previously unreported compounds (1–5, Fig. 1) are reported, as well as cytotoxic activities of all 58 isolates against A549, Colo205, QGY-7703, and THP-1 tumor cells.

2. Results and discussion The dried plant material of A. nephrolepis (50 kg) was extracted with 80% EtOH three times at 80 °C and the combined filtrates were concentrated in vacuo to a small volume, which was partitioned in successive steps using CHCl3, EtOAc and n-BuOH, respectively. The CHCl3 extract was separated into four fractions by column chromatography (CC) over silica gel. The EtOAc- and n-BuOH-soluble fractions were combined and three fractions were obtained by CC over macroporous absorption resin D101. These fractions were purified by repeated CC and Sephadex LH-20 CC, then subsequently by preparative TLC to afford compounds 1–5 and 53 known compounds. Compound 1 gave the molecular formula C10H16O3 by its positive HRESIMS at m/z 207.0995 [M+Na]+, indicating three degrees of unsaturation. The IR spectrum characteristically showed the presence of a carboxyl group (a broad band from 2500 to 3500, and 1707 cm1). The 13C and DEPT NMR spectra of 1 exhibited ten carbon signals, including two singlet methyls [dH 1.21 (3H, s, H3-9), 1.30 (3H, s, H3-8); dC 19.4 (C-9), 21.2 (C-8)], three methylenes [dC 22.9 (C-5), 23.3 (C-6), 35.9 (C-7)], two methines [dC 44.1

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8

21

4 5

7 1

2 9

3

1

19

11

18 17

14

10 4 2 2

20

29

5

7 30

28

26 27

Fig. 1. Compounds 1–6.

(C-1), 49.6 (C-4)], and three quaternary carbons including one carboxylic [dC 181.1 (C-10)], one oxygenated [dC 80.6 (C-3)], and one aliphatic quaternary groups [dC 57.2 (C-2)]. Based on the spin system of H-1/H-6a/H-5a/H-4/H-7/H-1 in the 1H–1H COSY spectrum (Fig. 2), a cyclopentane fragment was deduced. In the HMBC spectrum correlations originating from two methyls were found from dH 1.21 (H3-9) to dC 44.1 (C-1), 57.2 (C-2), 80.6 (C-3), 181.1 (C-10), and from dH 1.30 (H3-8) to dC 57.2 (C-2), 80.6 (C-3), 49.6 (C-4). In the ROESY spectrum correlations of H3-9 to H-5a/ H-6a/H3-8 and H-7a to H-5b/H-6b deduced the endo-orientation for H3-8 and H3-9 (Fig. 3). Therefore, the structure and relative stereochemistry of 1 were determined. However its absolute stereochemistry remained to be determined. Cu-Ka X-ray crystallographic analysis is commonly adopted to determine the absolute configuration (Chen and Cai, 2007; Hooft et al., 2008). Fortunately needle-shaped crystals of compound 1 were obtained in from methanol solution. Cu-Ka X-ray crystallographic analysis established unambiguously the absolute configuration of 1 as (1S,2R,3S,4R)-3-hydroxycamphane-2-carboxylic acid (Fig. 4). Interestingly, its stereosiomer, 3-endo-hydroxy-camphane-2-exo-carboxylic acid, had been discovered in racemic form in1969 (Finch and Vaughan, 1969; Rodig and Sysko, 1971). Compound 2 had the molecular formula C10H18O2 from its positive HRESIMS at m/z 193.1202 [M+Na]+, indicating two degrees of unsaturation. Comparison of its 13C NMR spectrum with that of 1 showed a close similarity except that the carboxyl group at dC 181.1 in 1 was replaced by a hydroxymethyl signal at dC 67.8 in 2. This deduction was confirmed by the HMBC correlation of H39 (dH 0.84) to C-10 (dC 67.8). By detailed analysis of its HSQC, COSY, HMBC, and ROESY spectra, compound 2 was identified as (1R,2S,3S,4S)-3-hydroxy-2-hydroxymethylcamphane. The molecular formula of compound 3 was determined as C16H28O7 by the positive HRESIMS at m/z 355.1740 [M+Na]+ (calcd for C16H28O7Na, 355.1733). The NMR spectroscopic data were

Fig. 2. Key 1H–1H COSY (bold) and HMBC (arrow) correlations of compound 1.

Fig. 3. Selected ROESY correlations of compound 1.

Fig. 4. X-ray (Cu-Ka) structure of compound 1.

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observed to be similar to those of 2, except for the presence of glycosyl group. By acid hydrolysis of 3, this was proved to be D-glucose, which was detected by TLC with an authentic sample, and the configuration determined by measurement of the optical rotation. In addition, the b-anomeric configuration of the glucose was indicated by its large coupling constant (3JH1,H2 = 7.9 Hz) (Sang et al., 2001). Based on the HMBC correlation of H-10 of the glucose to C-10, compound 3 was then assigned as (1R,2S,3S,4S)-3-hydroxy-2-hydroxymethylcamphane -10-O-b-D-glucopyranoside. The molecular formula of compound 4 was C30H42O4 by the positive HRESIMS at m/z 489.2988 [M+Na]+ (calcd for C30H42O4Na, 489.2981), indicating ten degrees of unsaturation. Its IR spectrum suggested the presence of hydroxyl (3442 cm1) and carbonyl groups (1762, 1703 cm1). Analysis of the 1H, 13C, and DEPT NMR spectroscopic data suggested the presence of five singlet and one doublet methyls [dH 0.89 (3H, s, H3-18), 1.05 (3H, s, H3-19), 1.02 (3H, d, J = 7.2 Hz, H3-21), 1.92 (3H, brs, H3-27), 1.10 (3H, s, H328), 1.07 (3H, s, H3-29); dC 18.6 (C-18), 18.6 (C-19), 16.9 (C-21), 10.4 (C-27), 26.5 (C-28), 21.1 (C-29)], ten methylenes including a terminal olefinic signal [dH 4.73, 4.47 (each for 1H, s, H2-30); dC 103.8 (t, C-30)], three methines with one from a vinyl group [dH 6.80 (1H, br s, H-24); dC 147.6 (d, C-24)], 11 quaternary carbons including one ketone [dC 217.6 (s, C-3)], one carbonyl [dC 171.7 (s, C-26)], four olefinic carbons [dC 136.3 (s, C-8), 147.8 (s, C-9), 131.5 (s, C-25), 155.1 (s, C-14)], and one oxygen-bearing quaternary signal [dC 106.7 (s, C-23)]. These data were in accordance with those of methyl (24Z)-8(14 ? 13)-abeo-17,13-friedo-lanosta8,14(30),24-triene-3,23-dion-26-oate (4a) (Raldugin et al., 1992), except that the side-chain in 4 is an epimeric mixture at the c-lactone moiety. This was consistent with the broad carbon resonances at dC 171.7 (s, C-26), 147.6 (d, C-24), 131.5 (s, C-25), 106.7 (s, C-23), 10.4 (q, C-27). Such tautomerism phenomena of lanostanes were reported before from other Abies species including Abies sachalinensis and Abies chensiensis (Gao et al., 2008; Li et al., 2009a). Since the circular dichroism (CD) spectrum of 4 showed a positive cotton effect (CE) at 288 nm and a negative CE at 233 nm, the absolute configurations of C-5 and C-20 were both deduced as R (Raldugin et al., 1992). Thus, compound 4 was elucidated as (5R,20R)-23-hydroxyl8(14 ? 13R)-abeo-17,13-friedo-3-oxolanosta-8,14(30),24-triene26,23-olide. A tentative biosynthetic pathway for formation of compound 4 is proposed in Fig. 5. It might be synthesized from neoabieslactone E (A) (Li et al., 2009a), via hydration to 17Shydroxyneoabieslactone E (B). Separate dehydration and dehydrogenation reaction of this intermediate would then provide

compound 4 (Kuroyanagi et al., 2000). It is interesting to note that compound 4 might have been isolated previously from the needles of A. sibirica (Raldugin et al., 1992). However this tautomeric compound was not characterized at that time. Instead, it was esterified with diazomethane to give its corresponding methyl ester (4a). Compound 5 exhibited a [M+Na]+ ion peak at m/z 497.3567 (calcd for C30H50O4Na, 497.3607) in the positive HRESIMS, corresponding to a molecular formula of C30H50O4. The NMR spectroscopic data of 5 were similar to those of 3-oxolanost-9bH-7-en24S,25-diol (Wada et al., 2002), except that the C-26 methyl group (dC 26.5 q) in 3-oxolanost-9bH-7-en-24S,25-diol was replaced by an oxygenated methylene (dC 68.9 t), suggesting a hydroxyl group located at C-26. Further confirmation was made by the HMBC correlation of H3-27 at dH 1.08 to C-26 at dC 68.9. Consequently, compound 5 was determined as 3-oxolanost-9bH-7-en-24,25,26-triol. By comparison of the NMR spectroscopic and MS data with the literature, 53 known compounds were identified as ten monoterpenoids: b-D-glucopyranosyl 2,3-endo-dimethyl-3-hydroxyl-10-norbornanoate (Yang et al., 2011), vicodiol (Vasanth et al., 1990), (E)-8-hydroxy-3,7-dimethyl-6-octenoic acid (Singh et al., 2000), (Z)-2,6-dimethyl-2,7-octadiene-1,6-diol (Knapp et al., 1998), (E)-2,6-dimethyl-2,7-octadiene-1,6-diol (Knapp et al., 1998), (+)-hydroxydihydroneocurueol (Diaz et al., 1994), uroterpenol (Miyazawa et al., 1998), angelicoidenol (Mahmood et al., 1983), (+)-angelicoidenol 2-O-b-D-glucopyranoside (Kitajima et al., 1998), ()-angelicoidenol 2-O-b-D-glucopyranoside (Kitajima et al., 1998), four apocarotenoids: tectoionol B (Macías et al., 2008), (6R,9S)-3-oxo-a-ionol b-D-glucopyranoside (Wang et al., 1998), blumenol C glucoside (Miyase et al., 1988), icarisides B5 (Miyase et al., 1988), nine sesquiterpenoids: ()-eudesm3b,4b,7a-triol (a new natural product) (Toyota et al., 1999), 3b,4b-dihydropallenone (Ahmed et al., 1990), aromadendrane4b,10b-diol (Wu et al., 2000), alismoxide (Yoshikawa et al., 1994), oplodiol (Feliciano et al., 1995), ent-T-muurolol (Nagashima et al., 1994), a-cadinol (6) (Borg-Karlson et al., 1981), 3b-hydroxya-cadinol (Lee and Chang, 2000), 3a-hydroxy-T-muurolol (Ding et al., 2009), one norditerpenoid: 8(14)-podocarpen-7,13-dien18-oic acid (Cheung et al., 1993), 15 diterpenoids: trans-phytol (7) (Sims and Pettus, 1976), (8a,12Z)-12,14-labdadien-8-ol (8) (Barrero and Altarejos, 1993), 13-epi-sclareol (9) (Torrenegra et al., 1992), sclareol (10) (Torrenegra et al., 1992), (12R,13E)8,12-epoxy-13-labden-15-ol (11) (Wahlberg et al., 1983), (12S,13E)-8,12-epoxy-13-labden-15-ol (12) (Wahlberg et al., 1983), (13E)-13-labdene-8a,15-diol (13) (Nishizawa et al., 1986),

2

Fig. 5. Postulated biosynthetic pathway for compound 4.

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abietic acid, 12-hyrdoxydehydroabietic acid (Kinouchi et al., 2000), 15-hyrdoxydehydroabietic acid (Cheung et al., 1993), isopimaric acid (Wenkert and Buckwalter, 1972), sandaracopimaric acids (Wenkert and Buckwalter, 1972), dehydroabietic acid, pomiferin A (Fraga et al., 1994), abiesadine D (Yang et al., 2010a), and fourteen triterpenoids: 21a-methoxyserrat-13-en-3-one (Tanaka et al., 1994),3-oxolanost-9bH-7-en-24S,25-diol (14) (Wada et al., 2002), 3-oxo-9bH-lanosta-7,22Z,24-trien-26,23-olide (15) (Kim et al., 2001), pindrolactone (Tripathi et al., 1996), 7,14,24-mariesatrien-26,23-olide-3a,23-diol (Gao et al., 2008), (24R)-cycloartane24,25-diol-3-one (16) (Inada et al., 1995), (24R)-cycloartane3a,24,25-triol (17) (Inada et al., 1997), (24R)-cycloartane3b,24,25-triol (18) (Inada et al., 1997), 24-methylene-3,4-secocycloart-4(28)-en-3-oic acid (Kim et al., 2001), 3b-methoxyserrat-14-en-21-one (Tanaka et al., 1994), 21a-methoxyserrat-14en-3-one (Tanaka et al., 1994), 23-oxo-mariesiic acid B (Hasegawa et al., 1987), 3b-methoxyserrat-14-en-21a-ol (Fang et al., 1991), (24R)-cycloartane-3b,24,25,28-tetrol (Inada et al., 1997). Among these compounds, 3b,4b-dihydropallenone is the first pallenone sesquiterpenoid isolated from the Pinaceae family. Meanwhile, aromadendrane sesquiterpenoid (aromadendrane-4b, 10b-diol) and serratane triterpenoids (21a-methoxyserrat-13-en3-one, 3b-methoxyserrat-14-en-21-one, 21a-methoxyserrat-14en-3-one, and 3b-methoxyserrat-14-en-21a-ol) are two types of chemical constituents obtained for the first time from Abies plants. All 58 isolates were tested for in vitro cytotoxic activities against A549, Colo205, QGY-7703, and THP-1 tumor cell lines (Table 3). Compounds 6, 7, 8, 9 + 10, 11 + 12, 13, and 18 demonstrated significant activities against A549 with IC50 values of 8.6, 11.5, 13.8, 10.9, 17.4, 11.5, and 17.3 lg/mL, respectively. While 6–9 + 10, 13, 14, 15, and 17 had significant inhibition of the viability of Colo205 with IC50 values of 8.1, 14.4, 12.5, 9.8, 11.3, 11.4, 13.6, and 19.4 lg/mL, respectively. For the QGY-7703 cell line, compounds 6–9 + 10, 11 + 12–14, and 18 showed strong inhibitory effects with IC50 values of 4.6, 12.2, 15.3, 7.0, 13.2, 6.8, 19.6, and 15.8 lg/mL, respectively. Only two mixtures of compounds 9 + 10 and 11 + 12 demonstrated significant inhibitory activities against THP-1 with IC50 values of 19.2 and 19.8 lg/mL, respectively. Although a-cadinol (6) exhibited the best cytotoxic effects on A549, Colo205 and QGY-7703 cells, its derivatives, 3b-hydroxy-acadinol, ent-T-muurolol, and 3a-hydroxy-T-muurolol, were negative. Comparison of their structures indicated that cadinane-type sesquiterpenoids with a hydroxyl group at C-3 and a trans-orien-

tated ring junction exhibited strongest cytotoxic activity. This is coincident with the order of antifungal index of the three compounds as a-cadinol > T-cadinol > T-muurolol (Chang et al., 2000) . Among 16 triterpenoids, 3-oxolanost-9bH-7-en-24S,25-diol (14), (24R)-cycloartane-24,25-diol-3-one (16), (24R)-cycloartane3a,24,25-triol (17), and (24R)-cycloartane-3b,24,25-triol (18) showed significant cytotoxic activities. This highlights the structure–activity relationship (SAR) importance of the alkyl side chain of 24,25-diol. 3. Conclusions From aerial parts of A. nephrolepis, 58 terpenoids with different skeletons were obtained including thirteen monoterpenoids, four apocarotenoids, nine sesquiterpenoids, 15 diterpenoids, one norditerpenoid, and 16 triterpenoids. Based on Cu-Ka X-ray crystallographic analysis and NMR spectroscopic methods, the structures of five new compounds were elucidated as three monoterpenoids: (1S,2R,3S,4R)-3-hydroxycamphane-2-carboxylic acid (1), (1R,2S,3S,4S)-3-hydroxy-2-hydroxymethylcamphane (2), (1R,2S,3S,4S)-3hydroxy-2-hydroxymethylcamphane-10-O-b-D-glucopyranoside (3), and two triterpenoids: (5R,20R)-23-hydroxyl-8(14 ? 13R)abeo-17,13-friedo-3-oxolanosta-8,14(30), 24-triene-26,23-olide (4), and 3-oxolanost-9bH-7-en-24,25,26-triol (5). All 58 isolates were tests for in vitro cytotoxic activity against four tumor cells of A549, Colo205, QGY-7703 and THP-1. Thirteen compounds (6– 18) exhibited positive effect, among which, a-cadinol (6) showed the best effects on A549, Colo205 and QGY-7703 with IC50 values of 8.6, 8.1 and 4.6 lg/mL, respectively. 4. Experimental 4.1. General experimental procedures Optical rotations were measured on a Perkin–Elmer 341 polarimeter, whereas UV and IR spectra were obtained by a Shimadzu UV-2550 spectrometer and a Bruker Vector 22 spectrometer with KBr pellets, respectively. NMR spectra were recorded on Bruker Avance 300, 400, or 600 NMR spectrometers with TMS as internal standard. ESIMS were acquired on an Agilent LC/MSD Trap XCT mass spectrometer, whereas HRESIMS were measured on a Waters Q-TOF micro mass spectrometer. Reversed phase medium pressure

Table 1 1 H and 13C NMR spectroscopic data (600 MHz) for compounds 1–3 (d in ppm, J in Hz). No.

1 (CDCl3)

2 (DMSO)

dC, mult

dH

dC, mult

dH

dC, mult

dH

1 2 3 4 5 6 7 8 9 10

44.1, CH 57.2, C 80.6, C 49.6, CH 22.9, CH2 23.3, CH2 35.9, CH2 21.2, CH3 19.4, CH3 181.1, C

2.53 br, s

43.6, 47.4, 79.5, 50.6, 22.7, 23.3, 34.0, 22.3, 18.4, 67.8,

1.73, d (1.8)

45.8, 48.8, 81.8, 51.8, 23.5, 24.3, 35.3, 22.7, 19.4, 79.3,

1.72, d (2.2)

2.07, 1.47, 1.47, 1.23, 1.30, 1.21,

d (3.6) 1.34, m 1.34, m d (10.2); 2.17, d (10.2) s s

CH C C CH CH2 CH2 CH2 CH3 CH3 CH2

3 (CD3OD)

1.83, 1.28, 1.44, 2.06, 1.08, 0.84, 3.17, 3.48,

d (4.2) m m,1.18, m d (9.6); 0.91, d (9.6) s s dd (10.5, 6.6); dd (10.5, 4.5)

10 20 30 40 50 60 OH (3) OH (10)

CH C C CH CH2 CH2 CH2 CH3 CH3 CH2

105.3, CH 74.9, CH 78.0, CH 71.5, CH 78.4, CH 62.7, CH2 4.18 br, s 4.14, dd (4.5, 6.6)

1.95, 1.37, 1.27, 1.07, 1.21, 1.01, 3.63,

d (4.1) m, 1.43, m m, 1.62, m d (9.9), 2.14, d (10.0) s s d (8.8), 3.74, d (8.6)

4.25, 3.15, 3.26, 3.28, 3.33, 3.66, 3.85,

d (7.9) dd (8.9, 8.0) m m m dd (11.9, 5.2); dd (12.0, 2.0)

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 OMe a b c d e

Recorded Recorded Recorded Recorded The NMR

4ae

4 (CDCl3)

5 (CD3OD)

dHa

dC, mult

dC, mult

dHc

dC, multd

1.62, 1.85, m 2.42, 2.58, m

35.6, CH2 34.5, CH2 217.6, C 47.2, C 51.0, CH 20.5, CH2 26.9, CH2 136.3, C 147.8, C 35.8, C 26.9, CH2 31.2, CH2 68.1, C 155.1, C 27.0, CH2 37.9, CH2 49.9, C 18.6, CH3 18.6, CH3 33.8, CH 16.9, CH3 39.3, CH2 106.7, C 147.6, CH 131.5, C 171.7, C 10.4, CH3 26.5, CH3 21.1, CH3 103.8, CH2

35.5, CH2 34.3, CH2 217.1, C 47.1, C 50.9, CH 20.5, CH2 26.3, CH2 136.1, C 147.8, C 35.8, C 26.4, CH2 32.4, CH2 68.0, C 155.5, C 27.1, CH2 37.7, CH2 49.1, C 18.6, CH3 18.5, CH3 34.1, CH 16.0, CH3 45.0, CH2 199.8, C 129.9, CH 141.2, C 169.4, C 20.1, CH3 26.5, CH3 20.9, CH3 104.0, CH2 52.1, CH3

1.64, m; 1.75, m 2.53, m

35.2, CH2 35.3, CH2 221.9, C 48.1, C 53.8, CH 23.9, CH2 122.7, CH 150.2, C 46.8, CH 37.0, C 21.9, CH2 35.7, CH2 45.1, C 53.1, C 34.3, CH2 29.3, CH2 54.5, CH 22.9, CH3 23.5, CH3 38.0, CH 19.1, CH3 34.6, CH2 28.8, CH2 77.3, CH 75.5, C 68.9, CH2 18.9, CH3 28.4, CH3 21.7, CH3 27.9, CH3

b

1.63, m 1.55, 1.65, m 1.93, 2.16, m

2.05, 2.12, m 1.40, 1.95, m

2.33, 2.37, m 1.45, 1.55, m 0.89, 1.05, 2.37, 1.02, 1.63,

s s m d (7.2) 2.26, m

6.80 br, s

1.92, 1.10, 1.07, 4.73,

s s s s; 4.47, s

d

1.47, m 1.84, 1.95, m 5.67, m 2.26, m 1.67, m 1.68, 1.89, m

1.43, 1.37, 1.59, 0.81, 0.99, 1.43, 0.93, 0.96, 1.14, 3.37,

1.59, m 2.02, m m s s m d (7.2) 1.81, m, 1.18, m d (11.4)

3.44, 1.08, 1.08, 1.08, 1.05,

d (11.1); 3.55, d (11.1) s s s s

at 300 MHz. at 75 MHz. at 400 MHz. at 100 MHz. data were taken from Raldugin et al. (1992).

Table 3 Cytotoxic effects of 13 compounds from A. nephrolepis against A549, Colo205, QGY-7703, and THP-1 cancer cell lines.a Compounds

IC50 (lg/mL) A549

Colo205

QGY-7703

THP-1

a-Cadinol (6)

8.6 11.5 13.8 10.9 17.4 11.5 >100.0 >100.0 >100.0 >100.0 17.3 1.0

8.1 14.4 12.5 9.8 21.5 11.3 11.4 13.6 27.4 19.4 33.3 0.8

4.6 12.2 15.3 7.0 13.2 6.8 19.6 >100.0 >100.0 26.1 15.8 0.6

>100.0 25.9 93.3 19.2 19.8 26.8 >100.0 >100.0 >100.0 >100.0 >100.0 1.0

trans-Phytol (7) (8a,12Z)-12,14-Labdadien-8-ol (8) 13-epi-Sclareol (9) + sclareol (10)b (12R,13E)-8,12-Epoxy-13-labden-15-ol (11) + (12S,13E)-8,12-Epoxy-13-labden-15-ol (12)b (13E)-13-Labdene-8a,15-diol (13) 3-Oxolanost-9bH-7-en-24S,25-diol (14) 3-Oxo-9bH-lanosta-7,22Z,24-trien-26,23-olide (15) (24R)-Cycloartane-24,25-diol-3-one (16) (24R)-Cycloartane-3a,24,25-triol (17) (24R)-Cycloartane-3b,24,25-triol (18) Doxorubicin hydrochloridec a b c

Fifty-eight isolates have been screened; inactive compounds (IC50 > 100 lg/mL) are not listed in this table. Mixture of two epimers (ca 1:1 for 9 and 10, while 3:5 for 11 and 12) were not subjected to further purification because of the low quantity. Positive control.

liquid chromatography (RP-MPLC) was performed on a Buchi Sepacore system (49  460 mm; m = 20 mL/min). Materials for column chromatography (CC) were silica gel (100–200 mesh; Huiyou Silica Gel Development Co. Ltd., Yantai, China), Sephadex LH-20 (40– 70 lm; Amersham Pharmacia Biotech AB, Uppsala, Sweden), and YMC-Gel ODS-A (50 lm; YMC, Milford, MA). Preparative TLC (20  20 cm; 0.4–0.5 mm) was conducted on glass plates precoated with silica gel GF254 (Yantai). Compounds were visualized by exposure to UV at 254 nm (Rf values range between 0.2 and 0.7).

4.2. Plant material Twigs, needles, and cones of A. nephrolepis from two trees were collected in August 2007 at an elevation of about 1300 m at longitude 128° East and latitude 42° North in Antu county, Jilin Province, China. The plant was authenticated by Prof. Han-Ming Zhang at the Second Military Medical University, China. A voucher specimen (No. 2007-08-003) was deposited in the Herbarium of School of Pharmacy at the Second Military Medical University.

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4.3. Extraction and isolation Dried plant material (50 kg) was pulverized and extracted three times with EtOH–H2O (80:20, V/V, 75 L) at 80 °C for 3 h. These three extracts were filtered through gauze, combined and concentrated to a small volume. The latter was partitioned sequentially with CHCl3 (60 L), EtOAc (20 L) and n-BuOH (20 L), respectively. The CHCl3 extract (2.5 kg) was separated into four fractions (C1–C4) by CC over silica gel eluting with a gradient of petroleum ether (PE, b.p. 60–90 °C)–CHCl3–EtOAc. Fraction C1 was subjected to CC over MCI, Sephadex LH-20 and silica gel to give ent-T-muurolol (10 mg), 6 (10 mg), 7 (63 mg), 8 (120 mg), abietic acid (69 mg), 21a-methoxyserrat-13-en-3-one (16 mg), and 15 (3 mg). Fraction C2 was divided into 6 subfractions (C2–1–C2–6) by CC over silica gel eluting with a gradient of PE-Me2CO (1:0 ? 0:1). Compounds 2 (24 mg), 14 (10 mg), and 16 (4 mg) were obtained from subfraction C2–2 after CC over Sephadex LH-20 (CHCl3–MeOH, 1:1), followed by repeated prep. TLC (CHCl3–MeOH, 30:1). From subfraction C2–3, 9 (4 mg), 10 (4 mg), 11 (4 mg), 12 (3 mg), and pindrolactone (40 mg) were isolated by MPLC eluting with MeOH–H2O (5:95 ? 100:0), followed by CC on Sephadex LH-20 (MeOH) and prep. TLC (CHCl3–MeOH, 20:1). Similarly, oplodiol (20 mg), 13 (200 mg), 12-hyrdoxydehydroabietic acid (7 mg), and 17 (10 mg) were obtained from subfractions C2–4, and 1 (50 mg), 15hyrdoxydehydroabietic acid (3 mg), 4 (10 mg), and 7,14,24-mariesatrien-26,23-olide-3a,23-diol (20 mg) from subfractions C2–5. Fraction C3 was separated into five subfractions (C3–1–C3–5) by CC over silica gel eluting with a gradient of PE-Me2CO (10:1 ? 2:1). Subfraction C3–3 was further subjected to CC over ODS (MeOH–H2O, 30:70 ? 100:0) and Sephadex LH-20 (MeOH). Final purification by prep. TLC (CHCl3–MeOH, 20:1) afforded (Z)-2,6-dimethyl-2,7-octadiene-1,6-diol (20 mg), (+)-hydroxydihydroneocurueol (50 mg), uroterpenol (4 mg), tectoionol B (4 mg), 3b,4b-dihydropallenone (32 mg), aromadendrane-4b,10b-diol (6 mg), alismoxide (7 mg), and 18 (17 mg). Similarly, (E)-8-hydroxy-3,7-dimethyl-6-octenoic acid (10 mg), (E)-2,6-dimethyl-2,7octadiene-1,6-diol (40 mg), angelicoidenol (38 mg), ()-eudesmane-3b,4b,7a-triol (10 mg), 3b-hydroxy-a-cadinol (10 mg), 8(14)-podocarpen-7,13-dien-18-oic acid (50 mg) were obtained from subfractions C3–4, and 3a-hydroxy-T-muurolol (28 mg) from subfraction C3–5. Fraction C4 was separated into three subfractions (C4–1–C4–3) by CC over silica gel eluting with a gradient of CHCl3– MeOH (100% ? 0%). Subfraction C4–1 was subjected to Sephadex LH-20 CC(CHCl3–MeOH, 1:1 and 0:1), followed by prep. TLC using PE-EtOAc (4:1) and CHCl3–MeOH (50:1) to give vicodiol (10 mg), isopimaric acid (14 mg), sandaracopimaric acid (65 mg), dehydroabietic acid (78 mg), pomiferin A (2 mg), 24-methylene-3,4-secocycloart-4(28)-en-3-oic acid (13 mg), 3b-methoxyserrat-14-en21-one (3 mg), 21a-methoxyserrat-14-en-3-one (2 mg), 3b-methoxyserrat-14-en-21a-ol (2 mg). By the same procedure, 5 (2 mg), abiesadine D (12 mg), 23-oxo-mariesiic acid B (16 mg), and (24R)cycloartane-3b,24,25,28-tetrol (3 mg) were obtained from subfraction C4–2. The EtOAc- and n-BuOH-soluble fractions were combined (2.13 kg) and separated into three fractions (E1–E3) by CC over macroporous absorption resin D101 eluting with EtOH–H2O (30:70, 70:30 and 95:5). The EtOH–H2O (30:70) eluate was collected and evaporated under vacuum to afford a residue (683 g). The latter was subjected to CC over silica gel eluting with a gradient of CHCl3–MeOH (5:1 ? 1:1) to give three subfractions (E1–1–E1–3). Subfraction E1–2 was further subjected to Sephadex LH-20 CC (MeOH), and repeated prep. TLC (CHCl3–MeOH, 10:1; EtOAc– MeOH–H2O, 20:3.4:2.7) to give (+)-angelicoidenol 2-O-b-D-glucopyranoside (36 mg), ()-angelicoidenol 2-O-b-D-glucopyranoside (25 mg), and icariside B5 (3 mg). Similarly, 3 (71 mg), b-D-glucopyranosyl 2,3-endo-dimethyl-3-hydroxyl-10-norbornanoate (38 mg),

(6R,9S)-3-oxo-a-ionol b-D-glucopyranoside (25 mg), and blumenol C glucoside (27 mg) were obtained from subfractions E1–3. 4.4. (1S,2R,3S,4R)-3-Hydroxycamphane-2-carboxylic acid (1) Colorless needles (MeOH); mp 120–122 °C; ½a20 D + 34.4 (c 0.22, MeOH); UV (MeOH) kmax (log e) 222 (1.73), 272 (0.76); IR (KBr) mmax 3404, 2960, 2878, 1723, 1707, 1691, 1487, 1383, 1263, 1129, 1058, 1034, 938, 900, 876, 789 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; ESI-MS (positive) m/z 207 [M+Na]+, 391 [2 M+Na]+; ESI-MS (negative) m/z 183 [MH]; HRESIMS (positive) m/z 207.0995 [M+Na]+ (calcd for C10H16O3Na, 207.0997). 4.5. (1R,2S,3S,4S)-3-Hydroxy-2-hydroxymethylcamphane (2) Colorless oil; ½a20 D + 11.6 (c 0.22, MeOH); IR (KBr) mmax 3423, 2962, 2933, 2879, 1701, 1636, 1465, 1384, 1113, 1024, 1009, 938, 915, 882, 840, 730 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; ESIMS (positive) m/z 193 [M+Na] +, 363 [2M+Na] +; ESIMS (negative) m/z 169 [MH], 339 [2 MH]; HRESIMS (positive) m/z 193.1202 [M+Na]+ (calcd for C10H18O2Na, 193.1204). 4.6. (1R,2S,3S,4S)-3-Hydroxy-2-hydroxymethylcamphane-10-O-b-Dglucopyranoside (3) Amorphous white powder; ½a23 D 6.6 (c 0.16, MeOH); IR (KBr) mmax 3416, 2959, 2929, 1708, 1630, 1383, 1159, 1076, 1034, 920, 631 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; ESIMS (positive) m/z 355 [M+Na]+, 687 [2M+Na]+; ESIMS (negative) m/z 367 [M+Cl], 663 [2 MH]; HRESIMS (positive) m/z 355.1740 [M+Na]+ (calcd for C16H25O7Na, 355.1733). 4.7. (5R,20R)-23-Hydroxyl-8(14 ? 13R)-abeo-17,13-friedo-3oxolanosta-8,14(30), 24-triene-26,23-olide (4) Amorphous white powder; ½a20 D + 6.6 (c 0.30, MeOH); CD (MeOH) De233 0.37, De289 + 0.36; UV (MeOH) kmax (log e) 217 (2.43); IR (KBr) mmax 3442, 2963, 1762, 1703, 1633, 1457, 1384, 1217,1113, 971, 882, 761 cm1; for 1H and 13C NMR spectroscopic data, see Table 2; ESIMS (positive) m/z 489 [M+Na]+; ESIMS (negative) m/z 465 [MH]; HRESIMS (positive) m/z 489.2988 [M+Na]+ (calcd for C30H42O4Na, 489.2981). 4.8. 3-Oxolanost-9bH-7-en-24,25,26-triol (5) Amorphous white powder; ½a20 D + 14.0 (c 0.09, MeOH); UV (MeOH) kmax (log e) 213 (3.41); IR (KBr) mmax 3400, 2933, 1702, 1604, 1515, 1456, 1376, 1271, 1209, 1142, 1116, 1033, 854, 816 cm1; for 1H and 13C NMR spectroscopic data, see Table 2; ESIMS (positive) m/z 497 [M+Na]+; ESIMS (negative) m/z 473 [MH]; HRESIMS (positive) m/z 497.3567 [M+Na]+ (calcd for C30H50O4Na, 497.3607). 4.9. X-ray data of compound 1 A colorless prism crystal was obtained from MeOH–H2O (5:1) at room temperature by slow evaporation. It had size 0.05  0.10  0.20 (mm), orthorhombic system, and space group P212121, Z = 4; a = 6.9477(7) Å, b = 12.9251(11) Å, c = 33.670(4) Å, V = 3032.6(11) Å3. Intensity data were collected on a Bruker SMART APEX-II diffractometer with graphite-monochromator at 296(2) K (Cu-Ka radiation, U/x scans). A total of 13,061 unique reflections were collected, of which 3630 were observed (|F|2 P 2r|F|2). The structures were solved by direct methods SHELXS-97 and refined

D.-W. Ou-Yang et al. / Phytochemistry 72 (2011) 2197–2204

with full-matrix least-squares on F2 using SHELEXL-97. The final refinement gave R1 = 0.0619 and xR2 = 0.1496 for observed data with I > 2r(I). Crystallographic data have been deposited with the Cambridge Crystallographic Data Center under CCDC 758806. These data can be obtained free of charge via www.ccdc.cam.ac.uk/ deposit (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; [email protected]). 4.10. Acid hydrolysis of compound 3 A solution of compound 3 (12.0 mg) in 6% aqueous HCl (5 mL) was heated until reflux began at 80 °C with this being maintained for 3 h. After cooling, the reaction mixture was diluted with H2O and then extracted with EtOAc. The aqueous layer was concentrated and the residue was subjected to silica gel CC with CHCl3– MeOH (6:4, V/V) as eluent to yield D-glucose (4.3 mg), ½a25 D + 65.0 (c 0.22, CH3OH). A monoterpene component isolated from the EtOAc fraction was identified as 2, by comparison of TLC and LC/ MS with the authentic sample. 4.11. Cell culture and cytotoxic assay A549 (human lung adenocarcinoma), Colo205 (colon adenocarcinoma), QGY-7703 (human hepatoma), and THP-1 (human monocytic leukemia) cell lines were obtained from the Shanghai Cell Bank, Chinese Academy of Sciences. The cytotoxic effects were measured in vitro using the MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] assay as previously reported (Yang et al., 2008c). Doxorubicin hydrochloride (Sigma; purity P 95%) was used as a positive control. Acknowledgment The work was supported by NCET Foundation, NSFC (30725045, 21002110), the Special Program for New Drug Innovation of the Ministry of Science and Technology, China (2009ZX09311-001, 2009ZX09308-005), Shanghai Leading Academic Discipline Project (B906) and in part by the Scientific Foundation of Shanghai, China (09DZ1975700, 09DZ1971500). Appendix A. Supplementary data Supplementary data associated with this article, including the 1D and 2D NMR spectra for compounds 1–5 as well as the chemical structures of the 53 known compounds, can be found in the online version, at doi:10.1016/j.phytochem.2011.08.003. References Ahmed, A.A., Jakupovic, J., Bohlmann, F., 1990. Dihydroxypallenone, a sesquiterpene with a new carbon skeleton from Pallenis spinosa. Phytochemistry 29, 3355– 3358. Barrero, A.F., Altarejos, J., 1993. 13C NMR data for labdane diterpenoids. Magn. Reson. Chem. 31, 299–308. Borg-Karlson, A.K., Norin, T., Talvitie, A., 1981. Configurations and conformations of torreyol (d-cadinol), a-cadinol, T-muurolol and T-cadinol. Tetrahedron 37, 425– 430. Chang, S.T., Wang, D.S., Wu, C.L., Shiah, S.G., Kuo, Y.H., Chang, C.J., 2000. Cytotoxicity of extractives from Taiwania cryptomerioides heartwood. Phytochemistry 55, 227–232. Chen, X.M., Cai, J.W., 2007. Principles and Practice of Single Crystal Structure Analysis, 2nd edition. Science Press, Beijing. Cheung, H.T.A., Miyase, T., Lenguyen, M.P., Smal, M.A., 1993. Further acidic constituents and neutral components of Pinus massoniana resin. Tetrahedron 49, 7903–7915. Diaz, J.G., Barba, B., Herz, W., 1994. Acetylenic and terpenoid constituents of Boltonia asteroides. Phytochemistry 36, 703–707. Ding, L., Pfoh, R., Ru, S., Qin, S., Laatsch, H., 2009. T-Muurolol sesquiterpenes from the marine streptomyces sp. M491 and revision of the configuration of previously reported amorphanes (1). J. Nat. Prod. 72, 99–101.

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