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Cytotoxic triterpenoids from Abies recurvata
Phytochemistry 81 (2012) 159–164
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Cytotoxic triterpenoids from Abies recurvata Yong-Li Li a, Yan-Xia Gao b, Xian-Wen Yang c,d,⇑, Hui-Zi Jin a, Ji Ye b, Luke Simmons c, Ning Wang c, Andre Steinmetz c, Wei-Dong Zhang a,b,⇑ a
School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China Department of Natural Product Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China c Cellular and Molecular Oncology Lab, Oncology Department, Luxembourg Public Research Center for Health (CRP-SANTE), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg d Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China b
a r t i c l e
i n f o
Article history: Received 17 February 2012 Received in revised form 15 May 2012 Available online 29 June 2012 Keywords: Abies recurvata Pinaceae Triterpenoids Cu-Ka X-ray crystallography Cytotoxicity
a b s t r a c t Nine triterpenoids (neoabiestrines A–I, 1–9) including six rearranged lanostanes (1–6) and a rare cycloart-7-ene (7) were isolated from Abies recurvata together with ten known compounds. Their structures were determined by detailed analysis of NMR and MS spectroscopic data. The absolute configurations of 1 and 8 were determined by Cu-Ka X-ray crystallography. Compound 6 showed potent anti-proliferative effect against THP-1 tumor cells with an IC50 value of 17.8 lg/mL. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Abies is an important genus of the Pinaceae family. There are ca 50 species around the world. Phytochemical studies on members of this genus revealed a variety of bioactive compounds with novel structures, with the main chemical constituents being triterpenoids (Gao et al., 2008a,b; Ou-Yang et al., 2011; Sun et al., 2008; Xia et al., 2012; Yang et al., 2008a, 2010). Abies recurvata is a woody plant distributed exclusively in China, especially in the north of Sichuan Province and the south of Gansu Province (Zheng and Fu, 1978). In the course of our discovery program for bioactive natural products, we identified this plant for further chemical and biological investigations, which resulted in the isolation of nine new (1– 9, Fig. 1) and 10 known (10–19) triterpenes belonging to lanostane and cycloartane structure classes. As the major triterpenoid in Abies plants, lanostanes were characterized as 18-Me and 30-Me located in C-14 and C-13 positions, respectively. Surprisingly, six lanostanes (1–6) isolated from A. recurvata were novel, with rear-
⇑ Corresponding authors at: Cellular and Molecular Oncology Lab, Oncology Department, Luxembourg Public Research Center for Health (CRP-SANTE), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg. Tel.: +352 26970 252; fax: +352 26970 390 (X.-W. Yang), Department of Natural Product Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China. Tel./ fax: +86 21 8187 1244 (W.-D. Zhang). E-mail addresses: [email protected] (X.-W. Yang), [email protected] (W.-D. Zhang). 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2012.05.032
ranged structures. Herein, we report the isolation, structural elucidation, and biological activity evaluation of these new and known triterpenes.
2. Results and discussion The CHCl3-soluble extracts of A. recurvata were subjected to column chromatography (CC) on silica gel, ODS, and Sephadex LH-20, and preparative TLC as well as preparative HPLC to afford 9 new and 10 known triterpenes. By comparison of their NMR and MS spectroscopic data with the literature, the known compounds were identified as cycloartane-3b,24,25-triol (10) (Inada et al., 1997), cycloartane-3a,24,25-triol (11) (Inada et al., 1997), abiesatrine I (12) (Yang et al., 2010), 3b-acetoxycycloart-24,25-diol (13) (Della Greca et al., 1994), neoabieslactone F (14) (Li et al., 2009), abiesatrine E (15) (Yang et al., 2010), firmanoic acid (16) (Hasegawa et al., 1987a), abiesatrine H (17) (Yang et al., 2010), abiesatrine B (18) (Yang et al., 2010), and 23-oxo-mariesiic acid B (19) (Hasegawa et al., 1987b). Compound 1 was obtained as colorless needles. The molecular formula was determined as C30H46O4 by the positive HRESIMS at m/z 493.3292 [M+Na]+. Its IR spectrum showed absorptions at 3422, 1741, and 1615 cm1, indicating the presence of hydroxyls, carbonyls, and olefinic bonds. The 1H, 13C, and DEPT NMR spectra of 1 exhibited thirty carbon signals comprising five singlet and two doublet methyls, eight methylenes, seven methines (one
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Fig. 1. Chemical structures of compounds 1–9.
Fig. 2. Key 1H–1H COSY, HMBC, and NOESY correlations for 1.
oxygenated and two olefinic ones), and eight quaternary carbons (two carbonyls and two olefinic ones). In the 1H–1H COSY spectrum, six fragments were established according to 1H–1H spin systems of H2-1/H2-2/H-3, H-5/H2-6/H-7, H-9/H2-11/H2-12, H-15/ H2-16, H3-21/H-20/H2-22, and H2-24/H-25/H3-27 (Fig. 2). Further HMBC correlations traced from seven methyls and two olefinic protons constructed the planar structure of 1 as 3-hydroxy-23oxomariesia-7,14-dien-26-oic acid (Fig. 2). Although the NOESY correlations could be used to deduce the relative stereochemistry of 1 (Fig. 2), the orientation of the carboxyl group at the C-26 position still remained unsolved. Fortunately, monoclinic crystals of 1 were obtained and subjected to the Cu-Ka X-ray crystallographic analysis, which established undoubtedly its absolute stereochemistry (Fig. 3). Accordingly, compound 1 was defined as (3R,25R)3-hydroxy-23-oxomariesia-7,14-dien-26-oic acid, and named neoabiestrine A. Compound 2 exhibited the molecular formula C31H48O4 as evidenced by the positive HRESIMS at m/z 507.3453 [M+Na]+. Its 1D NMR spectroscopic data were very similar to those of 1, except for an additional methoxyl moiety [dH 3.67 (3H, s, 26-OMe); dC 51.6 (q, 26-OMe)]. According to the HMBC correlation of 26-OMe to C-26 at dC 176.3, compound 2 was then elucidated as (3R,25R) methyl-3-hydroxy-23-oxomariesia-7,14-dien-26-oate, and named neoabiestrine B. Compound 3 showed the same molecular formula C31H48O4 based on its positive HRESIMS at m/z 507.3448 [M+Na]+. It displayed 1H and 13C NMR spectra similar to those of 19, except that the olefinic carbons located at the C-23 and C-24 positions in 19 were absent, while two sp3 signals (dC 34.6, 46.3) and an additional methoxyl moiety [dH 3.68 (3H, s, 26-OMe); dC 51.8 q]appeared. In the HMBC spectrum, correlations were found of H3-27 (dH 1.17, d, J = 7.2 Hz) to C-24 (dC 46.3 t), C-25 (dC 34.6 d), C-26 (dC 176.4 s), and of 26-OMe to C-26. Therefore, compound 3 was assigned
as (3R,25R) methyl-3-hydroxy-17,13-friedo-lanosta-23-oxo-7,12dien-26-oate, and named neoabiestrine C. Compound 4 was found to possess the molecular formula C30H44O4 from the positive HRESIMS at m/z 491.3135 [M+Na]+ (calcd. for C30H44O4Na, 491.3137). Its 1D NMR data were in accordance with those of (5R,20R,25R)-23-hydroxyl-8(14 ? 13R)abeo-17,13-friedo-3-oxolanosta-8,14(30),24-triene-26,23-olide (OuYang et al., 2011) except for the side chain which was consistent with that of 1. By detailed analysis of its HSQC, 1H–1H COSY, HMBC, and NOESY spectra, compound 4 was concluded as (13R,25R)8(14 ? 13)-abeo-17,13-friedo-lanosta-3,23-dion-8,14(30)-dien26-oic acid, and named neoabiestrine D. Compound 5 was assigned the molecular formula C31H46O4 from the positive HRESIMS at m/z 505.3297 [M+Na]+ (calcd. for C31H46O4Na, 505.3294). Its 1D NMR spectroscopic data were almost the same as those of 4, except for an additional methoxyl moiety [dH 3.65 (3H, s, 26-OMe); dC 51.8 (q, 26-OMe)]. In the HMBC spectrum, 26-OMe was correlated to C-26 (dC 176.1 s). Thus,
Fig. 3. X-ray structure of neoabiestrine A (1).
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Y.-L. Li et al. / Phytochemistry 81 (2012) 159–164 13
Fig. 4. X-ray structure of neoabiestrine H (8).
compound 5 was established as (13R,25R) methyl-8(14 ? 13)abeo-17,13-friedo-lanosta-3,23-dion-8,14(30)-dien-26-oate, and named neoabiestrine E. Compound 6 gave the molecular formula of C30H42O4 from the positive HRESIMS at m/z 489.2983 [M+Na]+ (calcd. for C30H42O4Na, 489.2981). Its NMR data were found to be very similar to those of 4, except that an olefinic bond was located at the C-24,C-25 positions. This was confirmed by the correlations of H3-26 (dH 2.14, d, J = 1.5 Hz) to C-24 (dC 132.1 d), C-25 (dC 145.0 s), and C-27 (dC 172.4 s). The downshifted chemical shifts of H-24 at dH 6.97 and H3-26 at dH 2.14 indicated that the vinyl bond presented should be E-orientated (Roshchin et al., 1987). Consequently compound 6 was determined as (24E,13R)-8(14 ? 13)-abeo-17,13-friedo-lanosta-3,23-dion-8,14(30),24-trien-27-oic acid, and named neoabiestrine F. Compound 7 had the same molecular formula of C30H42O4 with that of 1 on the basis of its positive HRESIMS at m/z 493.3296 [M+Na]+ (calcd. for C30H46O4Na, 493.3294). It also showed 1H and
C NMR spectra similar to those of 1, except that signals were found at dH –0.04 and 0.67 (J = 4.0 Hz, each for 1H) in 1H NMR spectrum and at dC (23.6 s, 25.0 s, and 24.7 t) in 13C NMR spectrum, which is characteristic for the cycloartane-type triterpene. By detailed analysis of its HSQC, 1H–1H COSY, HMBC, and NOESY spectra, compound 7 was then elucidated as 3a-hydroxy-cycloarta-23-on7-en-26b-oic acid, and named neoabiestrine G. Compound 8 had the molecular formula of C30H52O4 as established by its positive HRESIMS at m/z 499.3765 [M+Na]+ (calcd. for C30H52O4Na, 499.3763). It exhibited 1H and 13C NMR spectra very similar to those of 10, except for the presence of a hydroxy group at the C-28 position. This can be confirmed according to the HMBC correlations of the hydroxymethyl (dH 3.40, 3.58) to C3 (dC 77.1 d), C-4 (dC 43.6 s), C-5 (dC 37.4 d), and C-29 (dC 17.0 q), respectively. Further confirmation by Cu-Ka X-ray crystallographic analysis (Fig. 4) then established its absolute structure of compound 8 as (3R,24S)-cycloarta-3,24,25,28-tetraol, and named neoabiestrine H. Compound 9 gave a molecular formula C31H54O4 deduced from its positive HRESIMS at m/z 513.3923 [M+Na]+ (calcd. for C31H54O4Na, 513.3920). Its IR, NMR spectroscopic data were very similar to those of 8, except for an additional methoxyl moiety [dH 3.35 (3H, s); dC 56.5 (q)]. The HMBC correlation of the methoxyl to dC 86.0 implied that it was located at the C-3 position. Consequently compound 9 was determined as (3R,24S)-3-methoxycycloarta-24,25,28-triol, and named neoabiestrine I. To date compounds 1–19 represent the first triterpenoids reported from A. recurvata. Among them, 12 are lanostanes (1–6 and 14–19) and 7 are cycloartanes (7–13). These findings are consistent with molecular structure types reported previously from the genus of Abies and extend our knowledge of terpenoid metabolism.
Table 1 1 H NMR spectroscopic data for compounds 1–9 (J in Hz within parenthesis). No.
1a
1 2 3 5 6 7 8 9 11 12 15 16 17 18 19
0.89 1.57 3.39 1.60 1.93 5.56
m; 2.09 m m; 1.96 m brs m m; 1.98 m (d, 5.6)
0.94 1.56 3.45 1.54 1.87 5.56
m; 1.97 m m; 1.62 m brs m m; 1.96 m (dd, 1.4, 5.6)
1.00 1.63 3.46 1.48 1.91 5.64
m; 1.98 m m; 1.99 m brs m m (dd, 1.2, 5.6)
1.69 m; 1.73 m 1.67 m; 1.89 m 1.69 m; 1.92 m 1.04 2.38 m; 2.47 m 2.46 m; 2.55 m 2.24 m; 2.59 m 1.54 3.37 1.71 m 1.64 m 1.68 m 1.38 1.62 m; 1.69 m 1.58 m; 1.68 m 1.70 m 1.86 1.95 m; 2.05 m 2.01 m; 2.17 m 2.01 m; 2.25 m 5.42
1.39 1.58 1.50 5.16 1.90
m m; 1.83 m m; 1.86 m (dd, 1.2, 2.8) m; 2.20 m
1.37 1.38 1.40 5.18 1.88
m m; 1.77 m m; 1.82 m (dd, 1.4, 2.8) m; 2.18 m
1.43 1.82 5.46 1.42 1.93
m m; 2.23 m (dd, 2.0, 10.0) m; 1.53 m m
1.95 1.42 2.38 1.48
0.90 s 0.97 s
0.86 s 0.96 s
0.94 s 0.93 s
0.91 s 1.07 s
20 21 22
2.42 m 0.81 (d, 6.8) 2.30 m; 2.47 m
2.42 m 0.82 (d, 6.8) 2.74 m; 2.83 m
2.20 m 0.88 (d, 6.5) 2.74 m; 2.83 m
2.59 m 2.36 m 2.40 0.83 (d, 6.4) 0.82 (d, 6.4) 0.88 2.35 m; 2.70 m 2.20 m; 2.65 m 2.38 2.92
2.57 m; 2.82 m 2.85 m
2.20 m; 2.48 m 2.93 m
2.20 m; 2.48 m 2.93 m
2.46 m; 2.61 m 2.39 m; 2.77 m 6.97 (d, 1.5) 2.82 m 2.93 m
1.16 (d, 7.2) 0.96 s
1.16 (d, 7.2) 0.97 s
1.17 (d, 7.2) 0.98 s
1.13 (d, 7.2) 1.09 s
1.13 (d, 7.2) 1.09 s
2.14 (d, 1.5) 1.10 s
0.93 s 0.88 s
0.92 s 0.87 s
0.93 s 1.18 s
1.07 s 4.55 (d, 1.2) 4.76 (d, 1.2)
1.06 s 4.47 (d, 1.2) 4.72 (d, 1.2)
1.06 s 4.56 (d, 1.5) 4.76 (d, 1.5)
3.67 s
3.68 s
23 24 25 26 27 28 29 30 OMe a b c
2b
3b
Measured at 400 MHz in CD3OD. Measured at 400 MHz in CDCl3. Measured at 500 MHz in CD3OD.
4a
5a
m; 2.05 m 1.96 m m; 2.02 m 1.39 m; 1.96 m m 2.35 m m; 1.55 m 1.45 m; 1.53 m 0.86 s 1.02 s
3.65 s
6c
2.01 1.43 2.36 1.50 0.93 1.03
7a
8a
9a
m; 2.04 m 1.05 m; 1.91 m m; 1.91 m 1.59 m; 1.84 m (t, 2.4) 3.73 brs m 2.20 (dd, 4.0, 12.4) m 0.81 m; 1.50 m brs 1.16 m; 1.31 m 1.58 m
m; 2.25 m 1.28 m m; 2.03 m 1.09 m m 1.28 m m; 1.57 m 1.50 m; 1.85 m 2.57 m s 0.94 s s –0.04 (d, 4.2) 0.67 (d, 4.2) m 1.72 m (d, 6.5) 0.94 (d, 6.4) (d, 14.4); 2.28 m; 2.56 m (d, 14.4)
1.18 1.67 1.32 1.74 1.64 1.02 0.38 0.54 1.42 0.92 0.97
1.08 2.56 m; 2.83 m 3.16 2.80 m 1.12 1.14 (d, 7.2) 1.16 0.94 s 3.40 3.58 0.90 s 0.78 1.08 s 0.96
m; 2.05 m m m m; 1.95 m m s (d, 4.0) (d, 4.0) m (d, 6.4) m; 1.81 m
0.99 1.63 3.26 2.06 0.80 1.30 1.56
m; 1.75 m m; 1.90 m brs (dd, 4.4, 12.8) m; 1.45 m m; 1.12 m m
1.18 1.65 1.30 1.33 1.61 1.01 0.38 0.56 1.42 0.91 0.97
m; 2.00 m m m m; 1.62 m m s (d, 4.0) (d, 4.0) m (d, 6.4) m; 1.80 m
m; 1.34 m 1.34 m; 1.94 m (dd, 1.2, 10.0) 3.16 (dd, 1.6, 10.0) s s (d, 10.8) (d, 10.8) s s
1.12 1.16 3.38 3.52 0.84 0.95
s s (d, 10.8) (d, 10.8) s s
3.35 s
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Compounds 1–5 are novel lanosta-23-oxo-26-oic acid, which were rare in Abies species, with only three compounds reported in Abies marocana (Barrero et al., 1991), Abies alba (Leibyuk et al., 1990), and Abies sachalinensis (Wada et al., 2002). Interestingly, such lanostane-type compounds were usually found from the fungi Ganoderma lucidum (Cheng et al., 2010; Lee et al., 2010; Liu et al., 2006, 2009; Ma et al., 2002), Ganoderma applanatum (de Silva et al., 2006), Ganoderma sinense (Qiao et al., 2007), Daedaleopsis confragosa var. tricolor (Rösecke and König, 2000), Daedalea quercina (Polyporaceae) (Rösecke and König, 2000), Garcinia hombroniana (Rukachaisirikul et al., 2000, 2005), and Garcinia speciosa (Guttiferae) (Vieira et al., 2004). Compound 7 is a D7-cycloartane, reported from this genus for first time, previously reported from Cimicifuga simplex (Kusano et al., 1999a, 1996, 1998a,b, 1999b), Cimicifuga acerina (Kusano et al., 1998b), Cimicifuga heracleifolia (Li et al., 1993), Cimicifuga foetida (Kadota et al., 1995; Li et al., 1994), Cimicifuga rhizome (Koeda et al., 1995) , and Nigella sativa (Ranunculaceae) (Mehta et al., 2009, 2008). Thus, compounds 1–5 and 7 might be of chemotaxonomic significance for this species. Since triterpenoids from some Abies plants showed potent antitumor effects, all the isolates (1–19) were then subjected to in vitro bioassay for activity against THP-1, QGY-7703, and Colo205 cancer cells. Compound 6 showed positive effects with IC50 values of 17.8, 60.2, and 61.2 lg/mL, respectively.
28-hydroxyl-cycloartanes. Interestingly, these two types’ structures are found from this genus for the first time. This report adds to the current list of triterpenoids isolated from the genus of Abies. While initial evaluation of the anti-tumor efficacy of 1–19 is inconclusive, additional bio-assays are on going for a variety of human disease models.
3. Conclusions
4.2. Plant material
Lanostane and cycloartane triterpenoids are the primary chemical constituents of Abies species. This is coincident with the results obtained from this study on A. recurvata. Among the new compounds, 1–6 are novel rearranged lanostanes. In addition, 7 is a rarely found D7-cycloartane, and compounds 8 as well as 9 are
The aerial parts of A. recurvata were collected from Ya’an city, Sichuan Province of China in August 2009, and were identified by Prof. Han-Ming Zhang in Second Military Medical University. A herbarium specimen (No. 2009-08-001) was deposited in School of Pharmacy, Second Military Medical University, China.
4. Experimental 4.1. General experimental procedures NMR spectra were recorded on a Bruker Avance 400 or 500 NMR spectrometer with TMS as the internal standard. ESIMS were measured on an Agilent LC/MSD Trap XCT spectrometer, and HRESIMS were measured on an Agilent 6520 Accurate-Mass Q-TOF LCMS system. Optical rotations were acquired with Perkin-Elmer 341 polarimeter. IR spectra were recorded on a Bruker Vector-22 spectrometer with KBr pellets. Materials for CC were silica gel (Huiyou Silical Gel Development Co. Ltd., Yantai, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and YMCGEL ODS-A (YMC, USA). Reversed phase medium pressure liquid chromatography (RP-MPLC) was performed with a Buchi Sepacore system (49 460 mm; m = 20 mL/min). Preparative TLC was conducted with glass precoated silica gel GF254 (Yantai).
Table 2 13 C NMR spectroscopic data for compounds 1–9. No.
1a
2b
3b
4a
5a
6c
7a
8a
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
30.0 t 26.4 t 77.3 d 38.1 s 39.3 d 24.1 t 121.9 d 138.2 s 54.6 d 35.9 s 26.4 t 34.7 t 52.8 s 154.5 s 115.9 d 46.1 t 51.6 s 17.5 q 22.8 q 35.1 d 16.8 q 47.6 t 211.5 s 47.6 t 36.1 d 179.6 s 17.5 q 28.9 q 23.6 q 19.7 q
28.7 t 25.2 t 76.5 d 37.0 s 37.9 d 23.0 t 120.7 d 136.6 s 52.9 d 34.7 s 25.3 t 33.5 t 51.6 s 152.8 s 115.0 d 44.9 t 50.4 s 17.0 q 22.2 q 34.0 d 16.4 q 46.6 t 208.9 s 46.7 t 34.6 d 176.3 s 17.0 q 28.2 q 22.9 q 19.2 q
29.3 t 25.3 t 76.6 d 37.0 s 38.0 d 22.7 t 118.7 d 146.0 s 50.9 d 34.8 s 28.1 t 122.2 d 156.1 s 50.0 s 36.8 t 38.3 t 46.3 s 24.8 q 22.1 q 38.5 d 15.7 q 46.6 t 209.2 s 46.3 t 34.6 d 176.4 s 17.1 q 28.1 q 23.0 q 25.8 q
36.7 t 35.3 t 220.0 s 48.3 s 52.3 d 21.6 t 27.4 t 137.7 s 149.1 s 37.1 s 27.6 t 33.6 t 69.5 s 156.8 s 28.2 t 39.0 t 50.3 s 19.2 q 19.2 q 35.4 d 16.5 q 46.2 t 211.5 s 47.7 t 36.2 d 179.6 s 17.5 q 27.1 q 21.5 q 104.8 t
35.5 t 34.3 t 217.3 s 47.1 s 50.9 d 20.5 t 26.3 t 136.2 s 147.7 s 35.8 s 26.4 t 32.4 t 68.0 s 155.4 s 27.1 t 37.8 t 49.0 s 18.6 q 18.6 q 34.1 d 16.0 q 45.1 t 208.9 s 46.5 t 34.5 d 176.1 s 17.0 q 26.5 q 21.0 q 104.0 t
36.7 t 35.3 t 220.0 s 48.3 s 52.3 d 21.6 t 27.6 t 137.5 s 149.3 s 37.2 s 27.5 t 33.7 t 69.6 s 156.9 s 28.1 t 38.9 t 50.5 s 19.3 q 19.0 q 35.4 d 16.6 q 48.2 t 204.4 s 132.1 d 145.0 s 15.2 q 172.4 s 27.1 q 21.5 q 104.8 t
30.5 t 26.3 t 77.2 d 37.7 s 39.8 d 24.2 t 120.9 d 149.8 s 23.5 s 24.9 s 32.9 t 33.2 t 38.0 s 47.7 s 33.2 t 28.0 t 49.8 d 23.6 q 24.6 t 34.2 d 17.7 q 47.7 t 212.6 s 47.7 t 36.0 d 180.5 s 17.6 q 29.0 q 23.9 q 29.8 q
28.4 29.9 77.1 43.6 37.4 21.9 26.7 49.5 20.9 27.2 27.4 34.2 46.4 50.1 36.7 29.1 53.7 18.6 30.8 37.8 19.0 34.9 29.2 80.6 73.9 24.8 25.8 70.6 17.0 19.8
51.8 q
51.8 q
OMe a b c
Measured at 100 MHz in CD3OD. Measured at 100 MHz in CDCl3. Measured at 125 MHz in CD3OD.
51.8 q
9a t t d s d t t d s s t t s s t t d q t d q t t d s q q t q q
28.4 23.8 86.0 44.4 38.8 21.9 26.6 49.3 20.9 27.2 27.4 34.2 46.4 50.1 36.6 29.1 53.7 18.6 30.7 37.8 19.0 34.9 29.2 80.6 73.9 24.9 25.7 70.2 17.1 19.8
t t d s d t t d s s t t s s t t d q t d q t t d s q q t q q
56.5 q
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4.3. Extraction and isolation The plant material (17 kg) was pulverized and extracted with 95% EtOH under reflux for 3 3 h. The extracts were combined to concentrate to a small volume and then partitioned with CHCl3 (20 L) and EtOAc (20 L). The CHCl3 extract (1 kg) was separated into six fractions (C1–C6) by column chromatography (CC) over silica gel eluting with gradient petroleum ether (PE)-Me2CO. Fraction C1 (50 g) was subjected to CC over RP-MPLC, Sephadex LH-20, and silica gel to give 6 (12 mg). Fraction C2 was CC on silica gel with CHCl3–MeOH to yield two subfractions (C2.1 and C2.2). Subfraction C2.1 was purified by Sephadex LH-20 eluting with MeOH and repeated prep. TLC with CHCl3–MeOH (30:1) to give 1 (15 mg), 4 (36 mg), and 9 (14 mg). Compounds 10 + 11 (25 mg) and 18 + 19 (32 mg) were obtained from subfraction C2.2 after CC over Sephadex LH-20 (CHCl3–MeOH 1:1) followed by repeated prep. TLC with PE-Me2CO (3:1). Fraction C3 was divided into 20 subfractions (C3.1– C3.10) by RP-MPLC eluting with MeOH–H2O (50:50–100:0). After CC on Sephadex LH-20 with CHCl3–MeOH (1:1) and MeOH, subfraction C3.10 was then subjected to prep. TLC using CHCl3–MeOH (20:1) to give 2 (12 mg), 3 (11 mg), and 5 (38 mg). By similar procedures, compounds 7 (13 mg), 8 (18 mg), 12 + 13 (16 mg), 14 (12 mg), 15 (45 mg), 16 (15 mg), and 17 (16 mg) were isolated from subfraction C3.1 and C3.2, respectively. 4.3.1. Neoabiestrine A (1) Colorless monoclinic crystals; ½a20 D + 30.0 (c 0.20, MeOH); IR (KBr) mmax 3422, 2841, 2070, 1742, 1616, 1382, 1366, 1233, 1058, 1015, 682 cm1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 493 [M+Na]+, 963 [2 M+Na]+; ESIMS (negative) m/z 469 [MH], 939 [2 MH]; HRESIMS (positive) [M+Na]+ m/z 493.3292, calcd. for C30H46O4Na, 493.3294. 4.3.2. Neoabiestrine B (2) Amorphous powder; ½a20 D + 25.9 (c 0.50, MeOH); IR (KBr) mmax 3421, 2966, 2924, 2854, 1737, 1715, 1651, 1383, 1212, 984 cm1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 507 [M+Na]+, ESIMS (negtive) m/z 483 [MH]; HRESIMS (positive) [M+Na]+ m/z 507.3453, calcd. for C31H48O4Na, 507.3450. 4.3.3. Neoabiestrine C (3) Amorphous powder; ½a20 D 69.0 (c 0.20, MeOH); IR (KBr) mmax 3422, 2965, 2924, 2852, 1737, 1650, 1620, 1383, 1213, 987 cm1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 507 [M+Na]+, ESIMS (negtive) m/z 483 [MH]; HRESIMS (positive) [M+Na]+ m/z 507.3448, calcd. for C31H48O4Na, 507.3450. 4.3.4. Neoabiestrine D (4) Amorphous powder; ½a20 D + 14.5 (c 0.50, MeOH); IR (KBr) mmax 3380, 2968, 2361, 1649, 1620, 1382, 1233, 718 cm1; for 1H and 13 C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 491 [M+Na]+, ESIMS (negtive) m/z 467 [MH]; HRESIMS (positive) [M+Na]+ m/z 491.3135, calcd. for C30H44O4Na, 491.3137. 4.3.5. Neoabiestrine E (5) Amorphous powder; ½a20 D + 3.0 (c 0.50, MeOH); IR (KBr) mmax 3380, 2968, 2361, 1736, 1617, 1382, 1212, 686 cm1; for 1H and 13 C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 505 [M+Na]+, ESIMS (negtive) m/z 481 [MH]; HRESIMS (positive) [M+Na]+ m/z 505.3297, calcd. for C31H46O4Na, 505.3294. 4.3.6. Neoabiestrine F (6) Amorphous powder; ½a20 D + 14.9 (c 0.50, MeOH); IR (KBr) mmax 3426, 2962, 1693, 1646, 1382, 1212, 1112, 1026 cm1; for 1H and 13 C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 489
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[M+Na]+, ESIMS (negtive) m/z 465 [MH]; HRESIMS (positive) [M+Na]+ m/z 489.2983, calcd. for C30H42O4Na, 489.2981.
4.3.7. Neoabiestrine G (7) Amorphous powder; ½a20 D 78.8.0 (c 0.43, MeOH); IR (KBr) mmax 3434, 2962, 1710, 1637, 1461, 1367, 1213, 1064, 987 cm1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 493 [M+Na]+, ESIMS (negtive) m/z 469 [MH]; HRESIMS (positive) [M+Na]+ m/z 493.3296, calcd. for C30H46O4Na, 493.3294.
4.3.8. Neoabiestrine H (8) Colorless monoclinic crystals; ½a20 D + 11.8 (c 0.50, MeOH); IR (KBr) mmax 3421, 2924, 2361, 1651, 1621, 1382, 1232, 670 cm1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 499 [M+Na]+, ESIMS (negtive) m/z 475 [MH]; HRESIMS (positive) [M+Na]+ m/z 499.3765, calcd. for C30H52O4Na, 499.3763.
4.3.9. Neoabiestrine I (9) Amorphous powder; ½a20 D + 2.0 (c 0.20, MeOH); IR (KBr) mmax 3445, 2926, 1651, 1620, 1382, 1081 cm1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 499 [M+Na]+, ESIMS (negtive) m/z 475 [MH]; HRESIMS (positive) [M+Na]+ m/z 513.3923, calcd. for C31H54O4Na, 513.3920.
4.4. Cytotoxic assay As previously reported, three tumor cell lines of THP-1, QGY7703, and Colo205 were seeded into 96-well plates for 24 h (Yang et al., 2008b). Then different concentrations of the tested were added and incubation continued for 48 h. The absorbance was determined via a MTT assay with a microplate reader at 570 nm.
4.5. X-ray data of neoabiestrine A (1) and H (8) Neoabiestrine A (1): Colorless monoclinic crystals of C30H46O41=4 MeOH. Space group C2, a = 25.3688(4) Å, a = 90°; b = 7.32870(10) Å, b = 99.8060(10)°; c = 15.0899(2) Å, c = 90°; V = 2764.53(7) Å3, Z = 4; crystal size 0.28 0.25 0.20 mm3. Intensity data were collected on a Bruker APEX-II CCD diffractometer with a graphite-monochromator at 133(2) K (Cu-Ka radiation, U/x scans). A total of 28,064 unique reflections was collected, of which 4426 were observed (|F|2 P 2r|F|2). The structure was solved by direct methods (SHELXS-97) and refined with full-matrix least-squares on F2 using SHELEXL-97. The final refinement gave R1 = 0.0322 and xR2 = 0.0866 for observed data with I > 2r(I). Neoabiestrine H (8): Colorless monoclinic crystals of C30H52O4. Space group P2(1), a = 14.3580(2) Å, a = 90°; b = 6.89920(10) Å, b = 102.2610(10)°; c = 15.0899(2) Å, c = 90°; V = 1454.20(4) Å3, Z = 2; crystal size 0.20 0.08 0.04 mm3. Intensity data were collected on a Bruker APEX-II CCD diffractometer with a graphitemonochromator at 133(2) K (Cu-Ka radiation, U/x scans). A total of 10,264 unique reflections was collected, of which 4089 were observed (|F|2 P 2r|F|2). The structure was solved by direct methods (SHELXS-97) and refined with full-matrix least-squares on F2 using SHELEXL-97. The final refinement gave R1 = 0.0374, and xR2 = 0.1093 for observed data with I > 2r(I). The above crystallographic data have been deposited with the Cambridge Crystallographic Data Center under CCDC 863295 for neoabiestrine A (1) and CCDC 876644 for neoabiestrine H (8). 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]).
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Cytotoxic triterpenoids from Abies recurvata Yong-Li Li a, Yan-Xia Gao b, Xian-Wen Yang c,d,⇑, Hui-Zi Jin a, Ji Ye b, Luke Simmons c, Ning Wang c, Andre Steinmetz c, Wei-Dong Zhang a,b,⇑ a
School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China Department of Natural Product Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China c Cellular and Molecular Oncology Lab, Oncology Department, Luxembourg Public Research Center for Health (CRP-SANTE), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg d Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China b
a r t i c l e
i n f o
Article history: Received 17 February 2012 Received in revised form 15 May 2012 Available online 29 June 2012 Keywords: Abies recurvata Pinaceae Triterpenoids Cu-Ka X-ray crystallography Cytotoxicity
a b s t r a c t Nine triterpenoids (neoabiestrines A–I, 1–9) including six rearranged lanostanes (1–6) and a rare cycloart-7-ene (7) were isolated from Abies recurvata together with ten known compounds. Their structures were determined by detailed analysis of NMR and MS spectroscopic data. The absolute configurations of 1 and 8 were determined by Cu-Ka X-ray crystallography. Compound 6 showed potent anti-proliferative effect against THP-1 tumor cells with an IC50 value of 17.8 lg/mL. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Abies is an important genus of the Pinaceae family. There are ca 50 species around the world. Phytochemical studies on members of this genus revealed a variety of bioactive compounds with novel structures, with the main chemical constituents being triterpenoids (Gao et al., 2008a,b; Ou-Yang et al., 2011; Sun et al., 2008; Xia et al., 2012; Yang et al., 2008a, 2010). Abies recurvata is a woody plant distributed exclusively in China, especially in the north of Sichuan Province and the south of Gansu Province (Zheng and Fu, 1978). In the course of our discovery program for bioactive natural products, we identified this plant for further chemical and biological investigations, which resulted in the isolation of nine new (1– 9, Fig. 1) and 10 known (10–19) triterpenes belonging to lanostane and cycloartane structure classes. As the major triterpenoid in Abies plants, lanostanes were characterized as 18-Me and 30-Me located in C-14 and C-13 positions, respectively. Surprisingly, six lanostanes (1–6) isolated from A. recurvata were novel, with rear-
⇑ Corresponding authors at: Cellular and Molecular Oncology Lab, Oncology Department, Luxembourg Public Research Center for Health (CRP-SANTE), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg. Tel.: +352 26970 252; fax: +352 26970 390 (X.-W. Yang), Department of Natural Product Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China. Tel./ fax: +86 21 8187 1244 (W.-D. Zhang). E-mail addresses: [email protected] (X.-W. Yang), [email protected] (W.-D. Zhang). 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2012.05.032
ranged structures. Herein, we report the isolation, structural elucidation, and biological activity evaluation of these new and known triterpenes.
2. Results and discussion The CHCl3-soluble extracts of A. recurvata were subjected to column chromatography (CC) on silica gel, ODS, and Sephadex LH-20, and preparative TLC as well as preparative HPLC to afford 9 new and 10 known triterpenes. By comparison of their NMR and MS spectroscopic data with the literature, the known compounds were identified as cycloartane-3b,24,25-triol (10) (Inada et al., 1997), cycloartane-3a,24,25-triol (11) (Inada et al., 1997), abiesatrine I (12) (Yang et al., 2010), 3b-acetoxycycloart-24,25-diol (13) (Della Greca et al., 1994), neoabieslactone F (14) (Li et al., 2009), abiesatrine E (15) (Yang et al., 2010), firmanoic acid (16) (Hasegawa et al., 1987a), abiesatrine H (17) (Yang et al., 2010), abiesatrine B (18) (Yang et al., 2010), and 23-oxo-mariesiic acid B (19) (Hasegawa et al., 1987b). Compound 1 was obtained as colorless needles. The molecular formula was determined as C30H46O4 by the positive HRESIMS at m/z 493.3292 [M+Na]+. Its IR spectrum showed absorptions at 3422, 1741, and 1615 cm1, indicating the presence of hydroxyls, carbonyls, and olefinic bonds. The 1H, 13C, and DEPT NMR spectra of 1 exhibited thirty carbon signals comprising five singlet and two doublet methyls, eight methylenes, seven methines (one
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Fig. 1. Chemical structures of compounds 1–9.
Fig. 2. Key 1H–1H COSY, HMBC, and NOESY correlations for 1.
oxygenated and two olefinic ones), and eight quaternary carbons (two carbonyls and two olefinic ones). In the 1H–1H COSY spectrum, six fragments were established according to 1H–1H spin systems of H2-1/H2-2/H-3, H-5/H2-6/H-7, H-9/H2-11/H2-12, H-15/ H2-16, H3-21/H-20/H2-22, and H2-24/H-25/H3-27 (Fig. 2). Further HMBC correlations traced from seven methyls and two olefinic protons constructed the planar structure of 1 as 3-hydroxy-23oxomariesia-7,14-dien-26-oic acid (Fig. 2). Although the NOESY correlations could be used to deduce the relative stereochemistry of 1 (Fig. 2), the orientation of the carboxyl group at the C-26 position still remained unsolved. Fortunately, monoclinic crystals of 1 were obtained and subjected to the Cu-Ka X-ray crystallographic analysis, which established undoubtedly its absolute stereochemistry (Fig. 3). Accordingly, compound 1 was defined as (3R,25R)3-hydroxy-23-oxomariesia-7,14-dien-26-oic acid, and named neoabiestrine A. Compound 2 exhibited the molecular formula C31H48O4 as evidenced by the positive HRESIMS at m/z 507.3453 [M+Na]+. Its 1D NMR spectroscopic data were very similar to those of 1, except for an additional methoxyl moiety [dH 3.67 (3H, s, 26-OMe); dC 51.6 (q, 26-OMe)]. According to the HMBC correlation of 26-OMe to C-26 at dC 176.3, compound 2 was then elucidated as (3R,25R) methyl-3-hydroxy-23-oxomariesia-7,14-dien-26-oate, and named neoabiestrine B. Compound 3 showed the same molecular formula C31H48O4 based on its positive HRESIMS at m/z 507.3448 [M+Na]+. It displayed 1H and 13C NMR spectra similar to those of 19, except that the olefinic carbons located at the C-23 and C-24 positions in 19 were absent, while two sp3 signals (dC 34.6, 46.3) and an additional methoxyl moiety [dH 3.68 (3H, s, 26-OMe); dC 51.8 q]appeared. In the HMBC spectrum, correlations were found of H3-27 (dH 1.17, d, J = 7.2 Hz) to C-24 (dC 46.3 t), C-25 (dC 34.6 d), C-26 (dC 176.4 s), and of 26-OMe to C-26. Therefore, compound 3 was assigned
as (3R,25R) methyl-3-hydroxy-17,13-friedo-lanosta-23-oxo-7,12dien-26-oate, and named neoabiestrine C. Compound 4 was found to possess the molecular formula C30H44O4 from the positive HRESIMS at m/z 491.3135 [M+Na]+ (calcd. for C30H44O4Na, 491.3137). Its 1D NMR data were in accordance with those of (5R,20R,25R)-23-hydroxyl-8(14 ? 13R)abeo-17,13-friedo-3-oxolanosta-8,14(30),24-triene-26,23-olide (OuYang et al., 2011) except for the side chain which was consistent with that of 1. By detailed analysis of its HSQC, 1H–1H COSY, HMBC, and NOESY spectra, compound 4 was concluded as (13R,25R)8(14 ? 13)-abeo-17,13-friedo-lanosta-3,23-dion-8,14(30)-dien26-oic acid, and named neoabiestrine D. Compound 5 was assigned the molecular formula C31H46O4 from the positive HRESIMS at m/z 505.3297 [M+Na]+ (calcd. for C31H46O4Na, 505.3294). Its 1D NMR spectroscopic data were almost the same as those of 4, except for an additional methoxyl moiety [dH 3.65 (3H, s, 26-OMe); dC 51.8 (q, 26-OMe)]. In the HMBC spectrum, 26-OMe was correlated to C-26 (dC 176.1 s). Thus,
Fig. 3. X-ray structure of neoabiestrine A (1).
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Fig. 4. X-ray structure of neoabiestrine H (8).
compound 5 was established as (13R,25R) methyl-8(14 ? 13)abeo-17,13-friedo-lanosta-3,23-dion-8,14(30)-dien-26-oate, and named neoabiestrine E. Compound 6 gave the molecular formula of C30H42O4 from the positive HRESIMS at m/z 489.2983 [M+Na]+ (calcd. for C30H42O4Na, 489.2981). Its NMR data were found to be very similar to those of 4, except that an olefinic bond was located at the C-24,C-25 positions. This was confirmed by the correlations of H3-26 (dH 2.14, d, J = 1.5 Hz) to C-24 (dC 132.1 d), C-25 (dC 145.0 s), and C-27 (dC 172.4 s). The downshifted chemical shifts of H-24 at dH 6.97 and H3-26 at dH 2.14 indicated that the vinyl bond presented should be E-orientated (Roshchin et al., 1987). Consequently compound 6 was determined as (24E,13R)-8(14 ? 13)-abeo-17,13-friedo-lanosta-3,23-dion-8,14(30),24-trien-27-oic acid, and named neoabiestrine F. Compound 7 had the same molecular formula of C30H42O4 with that of 1 on the basis of its positive HRESIMS at m/z 493.3296 [M+Na]+ (calcd. for C30H46O4Na, 493.3294). It also showed 1H and
C NMR spectra similar to those of 1, except that signals were found at dH –0.04 and 0.67 (J = 4.0 Hz, each for 1H) in 1H NMR spectrum and at dC (23.6 s, 25.0 s, and 24.7 t) in 13C NMR spectrum, which is characteristic for the cycloartane-type triterpene. By detailed analysis of its HSQC, 1H–1H COSY, HMBC, and NOESY spectra, compound 7 was then elucidated as 3a-hydroxy-cycloarta-23-on7-en-26b-oic acid, and named neoabiestrine G. Compound 8 had the molecular formula of C30H52O4 as established by its positive HRESIMS at m/z 499.3765 [M+Na]+ (calcd. for C30H52O4Na, 499.3763). It exhibited 1H and 13C NMR spectra very similar to those of 10, except for the presence of a hydroxy group at the C-28 position. This can be confirmed according to the HMBC correlations of the hydroxymethyl (dH 3.40, 3.58) to C3 (dC 77.1 d), C-4 (dC 43.6 s), C-5 (dC 37.4 d), and C-29 (dC 17.0 q), respectively. Further confirmation by Cu-Ka X-ray crystallographic analysis (Fig. 4) then established its absolute structure of compound 8 as (3R,24S)-cycloarta-3,24,25,28-tetraol, and named neoabiestrine H. Compound 9 gave a molecular formula C31H54O4 deduced from its positive HRESIMS at m/z 513.3923 [M+Na]+ (calcd. for C31H54O4Na, 513.3920). Its IR, NMR spectroscopic data were very similar to those of 8, except for an additional methoxyl moiety [dH 3.35 (3H, s); dC 56.5 (q)]. The HMBC correlation of the methoxyl to dC 86.0 implied that it was located at the C-3 position. Consequently compound 9 was determined as (3R,24S)-3-methoxycycloarta-24,25,28-triol, and named neoabiestrine I. To date compounds 1–19 represent the first triterpenoids reported from A. recurvata. Among them, 12 are lanostanes (1–6 and 14–19) and 7 are cycloartanes (7–13). These findings are consistent with molecular structure types reported previously from the genus of Abies and extend our knowledge of terpenoid metabolism.
Table 1 1 H NMR spectroscopic data for compounds 1–9 (J in Hz within parenthesis). No.
1a
1 2 3 5 6 7 8 9 11 12 15 16 17 18 19
0.89 1.57 3.39 1.60 1.93 5.56
m; 2.09 m m; 1.96 m brs m m; 1.98 m (d, 5.6)
0.94 1.56 3.45 1.54 1.87 5.56
m; 1.97 m m; 1.62 m brs m m; 1.96 m (dd, 1.4, 5.6)
1.00 1.63 3.46 1.48 1.91 5.64
m; 1.98 m m; 1.99 m brs m m (dd, 1.2, 5.6)
1.69 m; 1.73 m 1.67 m; 1.89 m 1.69 m; 1.92 m 1.04 2.38 m; 2.47 m 2.46 m; 2.55 m 2.24 m; 2.59 m 1.54 3.37 1.71 m 1.64 m 1.68 m 1.38 1.62 m; 1.69 m 1.58 m; 1.68 m 1.70 m 1.86 1.95 m; 2.05 m 2.01 m; 2.17 m 2.01 m; 2.25 m 5.42
1.39 1.58 1.50 5.16 1.90
m m; 1.83 m m; 1.86 m (dd, 1.2, 2.8) m; 2.20 m
1.37 1.38 1.40 5.18 1.88
m m; 1.77 m m; 1.82 m (dd, 1.4, 2.8) m; 2.18 m
1.43 1.82 5.46 1.42 1.93
m m; 2.23 m (dd, 2.0, 10.0) m; 1.53 m m
1.95 1.42 2.38 1.48
0.90 s 0.97 s
0.86 s 0.96 s
0.94 s 0.93 s
0.91 s 1.07 s
20 21 22
2.42 m 0.81 (d, 6.8) 2.30 m; 2.47 m
2.42 m 0.82 (d, 6.8) 2.74 m; 2.83 m
2.20 m 0.88 (d, 6.5) 2.74 m; 2.83 m
2.59 m 2.36 m 2.40 0.83 (d, 6.4) 0.82 (d, 6.4) 0.88 2.35 m; 2.70 m 2.20 m; 2.65 m 2.38 2.92
2.57 m; 2.82 m 2.85 m
2.20 m; 2.48 m 2.93 m
2.20 m; 2.48 m 2.93 m
2.46 m; 2.61 m 2.39 m; 2.77 m 6.97 (d, 1.5) 2.82 m 2.93 m
1.16 (d, 7.2) 0.96 s
1.16 (d, 7.2) 0.97 s
1.17 (d, 7.2) 0.98 s
1.13 (d, 7.2) 1.09 s
1.13 (d, 7.2) 1.09 s
2.14 (d, 1.5) 1.10 s
0.93 s 0.88 s
0.92 s 0.87 s
0.93 s 1.18 s
1.07 s 4.55 (d, 1.2) 4.76 (d, 1.2)
1.06 s 4.47 (d, 1.2) 4.72 (d, 1.2)
1.06 s 4.56 (d, 1.5) 4.76 (d, 1.5)
3.67 s
3.68 s
23 24 25 26 27 28 29 30 OMe a b c
2b
3b
Measured at 400 MHz in CD3OD. Measured at 400 MHz in CDCl3. Measured at 500 MHz in CD3OD.
4a
5a
m; 2.05 m 1.96 m m; 2.02 m 1.39 m; 1.96 m m 2.35 m m; 1.55 m 1.45 m; 1.53 m 0.86 s 1.02 s
3.65 s
6c
2.01 1.43 2.36 1.50 0.93 1.03
7a
8a
9a
m; 2.04 m 1.05 m; 1.91 m m; 1.91 m 1.59 m; 1.84 m (t, 2.4) 3.73 brs m 2.20 (dd, 4.0, 12.4) m 0.81 m; 1.50 m brs 1.16 m; 1.31 m 1.58 m
m; 2.25 m 1.28 m m; 2.03 m 1.09 m m 1.28 m m; 1.57 m 1.50 m; 1.85 m 2.57 m s 0.94 s s –0.04 (d, 4.2) 0.67 (d, 4.2) m 1.72 m (d, 6.5) 0.94 (d, 6.4) (d, 14.4); 2.28 m; 2.56 m (d, 14.4)
1.18 1.67 1.32 1.74 1.64 1.02 0.38 0.54 1.42 0.92 0.97
1.08 2.56 m; 2.83 m 3.16 2.80 m 1.12 1.14 (d, 7.2) 1.16 0.94 s 3.40 3.58 0.90 s 0.78 1.08 s 0.96
m; 2.05 m m m m; 1.95 m m s (d, 4.0) (d, 4.0) m (d, 6.4) m; 1.81 m
0.99 1.63 3.26 2.06 0.80 1.30 1.56
m; 1.75 m m; 1.90 m brs (dd, 4.4, 12.8) m; 1.45 m m; 1.12 m m
1.18 1.65 1.30 1.33 1.61 1.01 0.38 0.56 1.42 0.91 0.97
m; 2.00 m m m m; 1.62 m m s (d, 4.0) (d, 4.0) m (d, 6.4) m; 1.80 m
m; 1.34 m 1.34 m; 1.94 m (dd, 1.2, 10.0) 3.16 (dd, 1.6, 10.0) s s (d, 10.8) (d, 10.8) s s
1.12 1.16 3.38 3.52 0.84 0.95
s s (d, 10.8) (d, 10.8) s s
3.35 s
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Compounds 1–5 are novel lanosta-23-oxo-26-oic acid, which were rare in Abies species, with only three compounds reported in Abies marocana (Barrero et al., 1991), Abies alba (Leibyuk et al., 1990), and Abies sachalinensis (Wada et al., 2002). Interestingly, such lanostane-type compounds were usually found from the fungi Ganoderma lucidum (Cheng et al., 2010; Lee et al., 2010; Liu et al., 2006, 2009; Ma et al., 2002), Ganoderma applanatum (de Silva et al., 2006), Ganoderma sinense (Qiao et al., 2007), Daedaleopsis confragosa var. tricolor (Rösecke and König, 2000), Daedalea quercina (Polyporaceae) (Rösecke and König, 2000), Garcinia hombroniana (Rukachaisirikul et al., 2000, 2005), and Garcinia speciosa (Guttiferae) (Vieira et al., 2004). Compound 7 is a D7-cycloartane, reported from this genus for first time, previously reported from Cimicifuga simplex (Kusano et al., 1999a, 1996, 1998a,b, 1999b), Cimicifuga acerina (Kusano et al., 1998b), Cimicifuga heracleifolia (Li et al., 1993), Cimicifuga foetida (Kadota et al., 1995; Li et al., 1994), Cimicifuga rhizome (Koeda et al., 1995) , and Nigella sativa (Ranunculaceae) (Mehta et al., 2009, 2008). Thus, compounds 1–5 and 7 might be of chemotaxonomic significance for this species. Since triterpenoids from some Abies plants showed potent antitumor effects, all the isolates (1–19) were then subjected to in vitro bioassay for activity against THP-1, QGY-7703, and Colo205 cancer cells. Compound 6 showed positive effects with IC50 values of 17.8, 60.2, and 61.2 lg/mL, respectively.
28-hydroxyl-cycloartanes. Interestingly, these two types’ structures are found from this genus for the first time. This report adds to the current list of triterpenoids isolated from the genus of Abies. While initial evaluation of the anti-tumor efficacy of 1–19 is inconclusive, additional bio-assays are on going for a variety of human disease models.
3. Conclusions
4.2. Plant material
Lanostane and cycloartane triterpenoids are the primary chemical constituents of Abies species. This is coincident with the results obtained from this study on A. recurvata. Among the new compounds, 1–6 are novel rearranged lanostanes. In addition, 7 is a rarely found D7-cycloartane, and compounds 8 as well as 9 are
The aerial parts of A. recurvata were collected from Ya’an city, Sichuan Province of China in August 2009, and were identified by Prof. Han-Ming Zhang in Second Military Medical University. A herbarium specimen (No. 2009-08-001) was deposited in School of Pharmacy, Second Military Medical University, China.
4. Experimental 4.1. General experimental procedures NMR spectra were recorded on a Bruker Avance 400 or 500 NMR spectrometer with TMS as the internal standard. ESIMS were measured on an Agilent LC/MSD Trap XCT spectrometer, and HRESIMS were measured on an Agilent 6520 Accurate-Mass Q-TOF LCMS system. Optical rotations were acquired with Perkin-Elmer 341 polarimeter. IR spectra were recorded on a Bruker Vector-22 spectrometer with KBr pellets. Materials for CC were silica gel (Huiyou Silical Gel Development Co. Ltd., Yantai, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and YMCGEL ODS-A (YMC, USA). Reversed phase medium pressure liquid chromatography (RP-MPLC) was performed with a Buchi Sepacore system (49 460 mm; m = 20 mL/min). Preparative TLC was conducted with glass precoated silica gel GF254 (Yantai).
Table 2 13 C NMR spectroscopic data for compounds 1–9. No.
1a
2b
3b
4a
5a
6c
7a
8a
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
30.0 t 26.4 t 77.3 d 38.1 s 39.3 d 24.1 t 121.9 d 138.2 s 54.6 d 35.9 s 26.4 t 34.7 t 52.8 s 154.5 s 115.9 d 46.1 t 51.6 s 17.5 q 22.8 q 35.1 d 16.8 q 47.6 t 211.5 s 47.6 t 36.1 d 179.6 s 17.5 q 28.9 q 23.6 q 19.7 q
28.7 t 25.2 t 76.5 d 37.0 s 37.9 d 23.0 t 120.7 d 136.6 s 52.9 d 34.7 s 25.3 t 33.5 t 51.6 s 152.8 s 115.0 d 44.9 t 50.4 s 17.0 q 22.2 q 34.0 d 16.4 q 46.6 t 208.9 s 46.7 t 34.6 d 176.3 s 17.0 q 28.2 q 22.9 q 19.2 q
29.3 t 25.3 t 76.6 d 37.0 s 38.0 d 22.7 t 118.7 d 146.0 s 50.9 d 34.8 s 28.1 t 122.2 d 156.1 s 50.0 s 36.8 t 38.3 t 46.3 s 24.8 q 22.1 q 38.5 d 15.7 q 46.6 t 209.2 s 46.3 t 34.6 d 176.4 s 17.1 q 28.1 q 23.0 q 25.8 q
36.7 t 35.3 t 220.0 s 48.3 s 52.3 d 21.6 t 27.4 t 137.7 s 149.1 s 37.1 s 27.6 t 33.6 t 69.5 s 156.8 s 28.2 t 39.0 t 50.3 s 19.2 q 19.2 q 35.4 d 16.5 q 46.2 t 211.5 s 47.7 t 36.2 d 179.6 s 17.5 q 27.1 q 21.5 q 104.8 t
35.5 t 34.3 t 217.3 s 47.1 s 50.9 d 20.5 t 26.3 t 136.2 s 147.7 s 35.8 s 26.4 t 32.4 t 68.0 s 155.4 s 27.1 t 37.8 t 49.0 s 18.6 q 18.6 q 34.1 d 16.0 q 45.1 t 208.9 s 46.5 t 34.5 d 176.1 s 17.0 q 26.5 q 21.0 q 104.0 t
36.7 t 35.3 t 220.0 s 48.3 s 52.3 d 21.6 t 27.6 t 137.5 s 149.3 s 37.2 s 27.5 t 33.7 t 69.6 s 156.9 s 28.1 t 38.9 t 50.5 s 19.3 q 19.0 q 35.4 d 16.6 q 48.2 t 204.4 s 132.1 d 145.0 s 15.2 q 172.4 s 27.1 q 21.5 q 104.8 t
30.5 t 26.3 t 77.2 d 37.7 s 39.8 d 24.2 t 120.9 d 149.8 s 23.5 s 24.9 s 32.9 t 33.2 t 38.0 s 47.7 s 33.2 t 28.0 t 49.8 d 23.6 q 24.6 t 34.2 d 17.7 q 47.7 t 212.6 s 47.7 t 36.0 d 180.5 s 17.6 q 29.0 q 23.9 q 29.8 q
28.4 29.9 77.1 43.6 37.4 21.9 26.7 49.5 20.9 27.2 27.4 34.2 46.4 50.1 36.7 29.1 53.7 18.6 30.8 37.8 19.0 34.9 29.2 80.6 73.9 24.8 25.8 70.6 17.0 19.8
51.8 q
51.8 q
OMe a b c
Measured at 100 MHz in CD3OD. Measured at 100 MHz in CDCl3. Measured at 125 MHz in CD3OD.
51.8 q
9a t t d s d t t d s s t t s s t t d q t d q t t d s q q t q q
28.4 23.8 86.0 44.4 38.8 21.9 26.6 49.3 20.9 27.2 27.4 34.2 46.4 50.1 36.6 29.1 53.7 18.6 30.7 37.8 19.0 34.9 29.2 80.6 73.9 24.9 25.7 70.2 17.1 19.8
t t d s d t t d s s t t s s t t d q t d q t t d s q q t q q
56.5 q
Y.-L. Li et al. / Phytochemistry 81 (2012) 159–164
4.3. Extraction and isolation The plant material (17 kg) was pulverized and extracted with 95% EtOH under reflux for 3 3 h. The extracts were combined to concentrate to a small volume and then partitioned with CHCl3 (20 L) and EtOAc (20 L). The CHCl3 extract (1 kg) was separated into six fractions (C1–C6) by column chromatography (CC) over silica gel eluting with gradient petroleum ether (PE)-Me2CO. Fraction C1 (50 g) was subjected to CC over RP-MPLC, Sephadex LH-20, and silica gel to give 6 (12 mg). Fraction C2 was CC on silica gel with CHCl3–MeOH to yield two subfractions (C2.1 and C2.2). Subfraction C2.1 was purified by Sephadex LH-20 eluting with MeOH and repeated prep. TLC with CHCl3–MeOH (30:1) to give 1 (15 mg), 4 (36 mg), and 9 (14 mg). Compounds 10 + 11 (25 mg) and 18 + 19 (32 mg) were obtained from subfraction C2.2 after CC over Sephadex LH-20 (CHCl3–MeOH 1:1) followed by repeated prep. TLC with PE-Me2CO (3:1). Fraction C3 was divided into 20 subfractions (C3.1– C3.10) by RP-MPLC eluting with MeOH–H2O (50:50–100:0). After CC on Sephadex LH-20 with CHCl3–MeOH (1:1) and MeOH, subfraction C3.10 was then subjected to prep. TLC using CHCl3–MeOH (20:1) to give 2 (12 mg), 3 (11 mg), and 5 (38 mg). By similar procedures, compounds 7 (13 mg), 8 (18 mg), 12 + 13 (16 mg), 14 (12 mg), 15 (45 mg), 16 (15 mg), and 17 (16 mg) were isolated from subfraction C3.1 and C3.2, respectively. 4.3.1. Neoabiestrine A (1) Colorless monoclinic crystals; ½a20 D + 30.0 (c 0.20, MeOH); IR (KBr) mmax 3422, 2841, 2070, 1742, 1616, 1382, 1366, 1233, 1058, 1015, 682 cm1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 493 [M+Na]+, 963 [2 M+Na]+; ESIMS (negative) m/z 469 [MH], 939 [2 MH]; HRESIMS (positive) [M+Na]+ m/z 493.3292, calcd. for C30H46O4Na, 493.3294. 4.3.2. Neoabiestrine B (2) Amorphous powder; ½a20 D + 25.9 (c 0.50, MeOH); IR (KBr) mmax 3421, 2966, 2924, 2854, 1737, 1715, 1651, 1383, 1212, 984 cm1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 507 [M+Na]+, ESIMS (negtive) m/z 483 [MH]; HRESIMS (positive) [M+Na]+ m/z 507.3453, calcd. for C31H48O4Na, 507.3450. 4.3.3. Neoabiestrine C (3) Amorphous powder; ½a20 D 69.0 (c 0.20, MeOH); IR (KBr) mmax 3422, 2965, 2924, 2852, 1737, 1650, 1620, 1383, 1213, 987 cm1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 507 [M+Na]+, ESIMS (negtive) m/z 483 [MH]; HRESIMS (positive) [M+Na]+ m/z 507.3448, calcd. for C31H48O4Na, 507.3450. 4.3.4. Neoabiestrine D (4) Amorphous powder; ½a20 D + 14.5 (c 0.50, MeOH); IR (KBr) mmax 3380, 2968, 2361, 1649, 1620, 1382, 1233, 718 cm1; for 1H and 13 C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 491 [M+Na]+, ESIMS (negtive) m/z 467 [MH]; HRESIMS (positive) [M+Na]+ m/z 491.3135, calcd. for C30H44O4Na, 491.3137. 4.3.5. Neoabiestrine E (5) Amorphous powder; ½a20 D + 3.0 (c 0.50, MeOH); IR (KBr) mmax 3380, 2968, 2361, 1736, 1617, 1382, 1212, 686 cm1; for 1H and 13 C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 505 [M+Na]+, ESIMS (negtive) m/z 481 [MH]; HRESIMS (positive) [M+Na]+ m/z 505.3297, calcd. for C31H46O4Na, 505.3294. 4.3.6. Neoabiestrine F (6) Amorphous powder; ½a20 D + 14.9 (c 0.50, MeOH); IR (KBr) mmax 3426, 2962, 1693, 1646, 1382, 1212, 1112, 1026 cm1; for 1H and 13 C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 489
163
[M+Na]+, ESIMS (negtive) m/z 465 [MH]; HRESIMS (positive) [M+Na]+ m/z 489.2983, calcd. for C30H42O4Na, 489.2981.
4.3.7. Neoabiestrine G (7) Amorphous powder; ½a20 D 78.8.0 (c 0.43, MeOH); IR (KBr) mmax 3434, 2962, 1710, 1637, 1461, 1367, 1213, 1064, 987 cm1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 493 [M+Na]+, ESIMS (negtive) m/z 469 [MH]; HRESIMS (positive) [M+Na]+ m/z 493.3296, calcd. for C30H46O4Na, 493.3294.
4.3.8. Neoabiestrine H (8) Colorless monoclinic crystals; ½a20 D + 11.8 (c 0.50, MeOH); IR (KBr) mmax 3421, 2924, 2361, 1651, 1621, 1382, 1232, 670 cm1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 499 [M+Na]+, ESIMS (negtive) m/z 475 [MH]; HRESIMS (positive) [M+Na]+ m/z 499.3765, calcd. for C30H52O4Na, 499.3763.
4.3.9. Neoabiestrine I (9) Amorphous powder; ½a20 D + 2.0 (c 0.20, MeOH); IR (KBr) mmax 3445, 2926, 1651, 1620, 1382, 1081 cm1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS (positive) m/z 499 [M+Na]+, ESIMS (negtive) m/z 475 [MH]; HRESIMS (positive) [M+Na]+ m/z 513.3923, calcd. for C31H54O4Na, 513.3920.
4.4. Cytotoxic assay As previously reported, three tumor cell lines of THP-1, QGY7703, and Colo205 were seeded into 96-well plates for 24 h (Yang et al., 2008b). Then different concentrations of the tested were added and incubation continued for 48 h. The absorbance was determined via a MTT assay with a microplate reader at 570 nm.
4.5. X-ray data of neoabiestrine A (1) and H (8) Neoabiestrine A (1): Colorless monoclinic crystals of C30H46O41=4 MeOH. Space group C2, a = 25.3688(4) Å, a = 90°; b = 7.32870(10) Å, b = 99.8060(10)°; c = 15.0899(2) Å, c = 90°; V = 2764.53(7) Å3, Z = 4; crystal size 0.28 0.25 0.20 mm3. Intensity data were collected on a Bruker APEX-II CCD diffractometer with a graphite-monochromator at 133(2) K (Cu-Ka radiation, U/x scans). A total of 28,064 unique reflections was collected, of which 4426 were observed (|F|2 P 2r|F|2). The structure was solved by direct methods (SHELXS-97) and refined with full-matrix least-squares on F2 using SHELEXL-97. The final refinement gave R1 = 0.0322 and xR2 = 0.0866 for observed data with I > 2r(I). Neoabiestrine H (8): Colorless monoclinic crystals of C30H52O4. Space group P2(1), a = 14.3580(2) Å, a = 90°; b = 6.89920(10) Å, b = 102.2610(10)°; c = 15.0899(2) Å, c = 90°; V = 1454.20(4) Å3, Z = 2; crystal size 0.20 0.08 0.04 mm3. Intensity data were collected on a Bruker APEX-II CCD diffractometer with a graphitemonochromator at 133(2) K (Cu-Ka radiation, U/x scans). A total of 10,264 unique reflections was collected, of which 4089 were observed (|F|2 P 2r|F|2). The structure was solved by direct methods (SHELXS-97) and refined with full-matrix least-squares on F2 using SHELEXL-97. The final refinement gave R1 = 0.0374, and xR2 = 0.1093 for observed data with I > 2r(I). The above crystallographic data have been deposited with the Cambridge Crystallographic Data Center under CCDC 863295 for neoabiestrine A (1) and CCDC 876644 for neoabiestrine H (8). 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]).
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