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Chemical constituents of Abies nukiangensis
Phytochemistry 106 (2014) 116–123
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Chemical constituents of Abies nukiangensis Yong-Li Li a,d, Yan-Xia Gao a, Hui-Zi Jin a, Lei Shan b, Xue-Song Liang e,f, Xi-Ke Xu b, Xian-Wen Yang c,⇑, Ning Wang c, Andre Steinmetz c, Zhilei Chen f, Wei-Dong Zhang a,b,⇑ a
School of Pharmacy, Shanghai JiaoTong University, 800 Dongchuan Road, Shanghai 200240, China School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China c Luxembourg Public Research Centre for Health (CRP-Sante), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg d Division of Material Science & Quality Test, Shanghai Institute of Measurement and Testing Technology, Shanghai 201203, China e Department of Infectious Diseases, Changhai Hospital, Second Military Medical University, Shanghai 200433, China f Artie McFerrin Department of Chemical Engineering, Texas A&M University, TX 77843, USA b
a r t i c l e
i n f o
Article history: Received 9 April 2014 Received in revised form 1 July 2014 Available online 28 July 2014 Keywords: Abies nukiangensis Hepatitis C virus (HCV) Pinaceae Abietoideae Triterpenoids Cu-Ka X-ray crystallography
a b s t r a c t During a survey on chemical constituents of Abies nukiangensis, seven previously unreported compounds, including six triterpenes (1–6) and one phenol (7) were isolated and characterized, together with 37 known miscellaneous chemical constituents. The structures of compounds 1–7 were established mainly by extensive analysis of the 1D and 2D NMR, as well as HRMS data. The absolute configurations of compounds 1 and 8 were confirmed unambiguously by the Cu-Ka X-ray crystallography. Compounds 3 and 8–10 showed significant anti-hepatitis C virus effects with EC50 values of 3.73, 2.67, 1.33 and 2.25 lM, respectively. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Abies plants are a rich source to produce bioactively potent and structurally diverse chemical constituents (Kim et al., 2004; Lavoie et al., 2012; Li et al., 2012b; Wada et al., 2002; Yang et al., 2008a, 2009, 2010b). Since 2006, our research interest has focused on this genus, particularly on those occurring exclusively in Mainland China. Abies nukiangensis Cheng et L.K. Fu (Pinaceae) is a tall tree distributing solely in Mainland China, especially in the northwest areas of Guangxi Province (Zheng and Fu, 1978). As a continuing study, this plant was collected for systematic investigations. Consequently, seven new (1–7, Fig. 1) and 37 known compounds were obtained. Here we report the isolation and structure elucidation of the new compounds. The activities of all the isolates for anti-hepatitis C virus (anti-HCV) on n4mBid cells and cytotoxicity against QGY-7703 and A549 tumor cell lines were evaluated.
The CHCl3-soluble extract of A. nukiangensis was subjected to repeated column chromatography (CC) over silica gel, ODS, and Sephadex LH-20, followed by purification using preparative TLC to yield seven new and 37 known compounds. By comparison of their NMR and MS data with the published references, the known compounds were then identified as seven triterpenes: 3-oxolanost9(11)-en-24S,25-diol (8) (Wada et al., 2001), lanost-9(11)en-3a,24S,25-triol (9) (Ohtsu et al., 1998), neoabiestrine F (Li et al., 2012a), 23-hydroxyl-8(14?13R)-abeo-17,13-friedo-3-oxolanosta-8,14(30),24-triene-26,23-olide (Ou-Yang et al., 2011), isomangiferolic acid (Escobedo-Martínez et al., 2012), mangiferolic acid (Escobedo-Martínez et al., 2012), mangiferonic acid (Escobedo-Martínez et al., 2012); 17 diterpenes: aquilarabietic acid J (Yang et al., 2013), abietic acid (Liu et al., 2009), abieta-7,13dien-12a-methoxy-18-oic acid (Wu et al., 2010), dehydroabietic acid (Cheung et al., 1993), 15-hydroxydehydroabietic acid (Cheung et al., 1993), abiesadine N (Yang et al., 2010a), 7a-hydroxydehydroabietic acid (Cheung et al., 1993), 7b-hydroxydehydroabietic acid (Cheung et al., 1993), 18-succinoyloxyabieta-8,11, 13-triene (Raldugin et al., 2005), abiesadine P (Yang et al., 2010a), 7-oxodehydroabietinol (Tanaka et al., 1997), abiesadine I (Yang et al., 2010a), 9,13b-epidioxy-8(14)-abieten-18-oic acid
⇑ Corresponding authors at: Luxembourg Public Research Center for Health (CRPSante), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg. Tel.: +352 26970 393; fax: +352 26970 390 (X.-W. Yang), School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China. Tel./fax: +86 21 81871244 (W.-D. Zhang). E-mail addresses: [email protected] (X.-W. Yang), wdzhangy@hotmail. com (W.-D. Zhang). http://dx.doi.org/10.1016/j.phytochem.2014.07.003 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Y.-L. Li et al. / Phytochemistry 106 (2014) 116–123
117
Fig. 1. Chemical structures of compounds 1–8.
(Barrero et al., 1991), 13,15-dihydroxypodocarpa-8,11,13-triene (Georges et al., 2012), 7a-hydroxymanool (Jiang et al., 2001), 8-hydroxy-12-oxo-13-abieten-18-oic acid (Baltenweck-Guyot et al., 2000), trans-phytol (Sims et al., 1976); four sesquiterpenes: 4-[4-hydroxy-6-(hydroxymethyl)-2,6-dimethyl-1-cyclohex-1-en1-yl] butan-2-one (Miyase et al., 1987), clovane-2b,9a-diol(Heymann et al., 1994), caryolane-1,9b-diol (Heymann et al., 1994), 8,9-dihydroxy-1(12)-caryophllene (Heymann et al., 1994); three lignans: dihydrodehydrodiconiferyl alcohol (Silva et al., 1989), pinoresinol (Abe and Yamauchi, 1988), prinsepiol (Kilidhar et al., 1982); and six other compounds: 4-hydroxy-3-methoxycinnamaldehyde (Kikuzaki et al., 1999), 5,40 -dihydroxy-3,7-dimethoxy-6methylflavone (10) (Li et al., 2011), rhododendrol (Kubo et al., 1983), 4-(p-hydroxyphenyl)-2-butanone (Ayer and Singer, 1980), b-sitosterol (Hussain et al., 2008), and stigmastanone (Guerriero et al., 1993). Compound 1 was obtained as colorless needle crystals. The HRESIMS of 1 gave a pseudo-molecular ion peak at m/z 399.2900 [M+H]+, consistent with the molecular formula of C26H38O3. The IR spectrum indicated absorption bands of carbonyls (1706 cm1) and olefinic bonds (1646 cm1). The 1H-NMR spectrum of 1 exhibited resonances for four methyl singlets [dH 0.88 (3H, s), 1.07 (6H, s), 1.11 (3H, s)], one methyl doublet [dH 0.94 (3H, d, J = 6.5 Hz)], and one exomethylene [d 4.51 (1H, brs), 4.76 (1H, brs)]. The 13C-NMR spectrum, with the aid of the DEPT technique, resolved 26 carbon signals including five methyls, 10 methylenes (one olefinic), two methines, and nine quaternary carbons (two carbonyls and three olefinic ones) (Table 1). Considering the characteristic signals of one sp3 quaternary carbon (dC 68.1) and two sp2 olefinic bonds (dC 135.9 s, 148.0 s, 104.1 t, 155.4 s), compound 1 was deduced to be a lanostane triterpene containing a 8(14?13R) abeo-17, 13-friedo moiety (Li et al., 2012a; Ou-Yang et al., 2011). The whole planar structure of 1 were deduced by the 1H–1H COSY correlations of five isolated spin systems of H2-1/H2-2, H-5/H2-6/H2-7, H2-11/ H2-12, H2-30/H2-15/H2-16, and H3-21/H-20/H2-22, coupling with the HMBC correlations of Me-18, Me-19, Me-21, Me-28, Me-29, and H2-30 (Fig. 2). Its absolute stereochemistry was established undoubtedly by the Cu-Ka X-ray crystallographic analysis
(Fig. 3). Accordingly, compound 1 was determined unequivocally to be 24,25,26,27-tetranor-8(14?13R)abeo-17,13-friedo-3-oxolanosta-8,14(30)-dien-23-oic acid. Interestingly, compound 1 is the first example of the nortriterpene bearing a 8(14?13) abeo-17,13-friedo moiety. Compound 2 was assigned the molecular formula C30H42O3 from the positive HRESIMS at m/z 451.3208 [M+H]+. The IR spectrum showed the presence of a saturated carbonyl group (1714 cm1) and an a,b-unsaturated butyrolactone (1754 cm1). Its 1H- and 13C-NMR data were in accordance with those of 1 except that the carboxyl moiety at the C-23 position in 1 was substituted by an a,b-unsaturated butyrolactone in 2, which was deduced from the IR absorption (1754 cm1) and the 13C resonances [dC 79.3 (C-23), 149.6 (C-24), 129.3 (C-25), 174.1 (C-26), 10.5 (C-27)]. The assumption was confirmed by the HMBC correlations of H-24 (dH 4.92) to C-22, C-23, C-25, C-26, and C-27. In the CD spectrum, compound 2 exhibited a negative cotton effect at 212 nm (28.2), indicating a R-configuration of the C-23 position (Allen et al., 1971). On the basis of above evidences, 2 was therefore proposed as (13R,23R)-8(14?13)abeo-17,13-friedo-3oxolanosta-8,14(30),24-trien-26,23-olide. Compound 3 was determined to have the molecular formula C31H54O3 from the positive HRESIMS at m/z 497.3973 [M+Na]+. Its NMR spectroscopic data were very similar to those of 3-oxolanost-9(11)-en-24S,25-diol (8), except that the ketone group (dC 217.3) was replaced by an oxygenated methine (dC 85.9). Furthermore, an additional methoxy moiety [dH 3.31(3H, s); dC 57.0] was found, which indicated that a methoxy group was located at the C-3 position in 3 instead of a ketone moiety in 8. The assumption was confirmed by the correlations of H3-28 (dH 0.94), H3-29 (dH 0.88), and the methoxy (dH 3.31) to the oxygenated methine (dC 85.9) in the HMBC spectrum. The a-configuration of C-3 was established according to the small coupling constant of H-3 (dH 2.82, brs) (Yang et al., 2010b), which was further confirmed by the NOESY correlation of H-3 to H3-29 and H3-29 to H3-18. Interestingly, previously isolated lanostane triterpenes containing a 24,25-dihydroxy moiety had all 24S configuration determined either by the CD spectrum using 2% Eu(dpm)3 (Wada et al.,
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Y.-L. Li et al. / Phytochemistry 106 (2014) 116–123
Table 1 H-NMR data for compounds 1–6 and 8 in CDCl3 (J in Hz within parenthesis).
1
No.
1a
2a
3b
1 2 3 5 6 7 8 11 12
1.68 m, 1.90 m 2.49 m, 2.60 m
1.64 m, 1.90 m 2.43 m, 2.57 m
1.65 m 1.60 m, 1.69 m 1.95 m, 2.14 m
1.62 m 1.66 m 1.92 m, 2.12 m
1.95 m, 2.14 m 1.44 m, 2.06 m
1.92 m, 2.12 m 1.38 m, 2.12 m
m, 1.81 m m, 2.03 m brs m m, 1.59 m m,1.64 m m (brd, 5.0) m, 2.09 m
1.54 1.76 4.48 0.97 1.48 1.34 2.17 5.23 1.90
m, 1.79 m m (dd, 4.0, 11.5) m m, 1.68 m m,1.67 m (brd, 13.5) (d, 6.0) m, 2.08 m
2.34 m, 2.39 m 1.49 m, 1.56 m
2.33 m, 2.39 m 1.49 m, 1.54 m
0.88 1.07 2.33 0.94 1.99 2.74
0.88 0.92 2.25 0.99 1.38
m, 1.73 m m, 1.60 m brs m m, 1.86 m m,1.67 m m (d, 5.0) m, (brd, 14.0) m m, 1.93 m m s s m (d, 5.5) m, 1.77 m
1.51 1.69 3.44 1.32 1.49 1.34 2.20 5.26 1.90
15 16 17 18 19 20 21 22
1.25 1.39 2.82 1.30 1.72 1.34 2.17 5.23 1.89 2.09 1.34 1.64 1.61 0.65 1.06 1.41 0.90 1.00
1.37 1.64 1.62 0.66 1.07 1.41 0.90 1.27
m m,1.92 m m s s m (d, 7.5) m, 1.82 m
1.35 1.67 1.62 0.65 1.07 1.41 0.90 1.00
1.14 3.29 1.17 1.22 0.94 0.88 0.74 3.31
m, 1.60 m m s s s s s s
2.50 m, 2.56 m
1.13 3.29 1.17 1.22 0.87 0.89 0.74
23 24 26 27 28 29 30 OMe OAc OEt a b
s s m (d, 6.5) (brd, 14.5), (brd, 14.5)
s s m (d, 6.5) m, 1.73 m
4.92 (d, 11.0) 7.01 s
1.11 s 1.07 s 4.51 brs, 4.76 brs
1.87 1.09 1.04 4.46
s s s brs, 4.72 brs
4a
1.39 1.39 0.97 0.89 0.77
5a
s s s s s
6a
8a m, 1.82 m m (dd, 4.0, 12.0) m m, 1.70 m m, 1.68m (brd, 12.0) (d, 6.0) m, (brd, 17.0) m, 1.86 m m m s s m (d, 6.5) m, 1.86 m
1.37 1.63 1.35 2.22 5.28 1.93
m m m, 1.70 m (brd, 12.5) (d, 5.5) m, 2.09 m
m m, 1.91 m m s s m (d, 7.5) m, 1.78 m
1.44 1.75 3.22 0.89 1.50 1.36 2.17 5.23 1.89 2.09 1.37 1.31 1.62 0.66 1.05 1.44 0.90 1.37
1.81 m, 2.10 m 2.40 m, 2.72 m
1.37 1.36 1.62 0.67 1.22 1.42 0.90 1.00
m m, 1.93 m m s s m (d, 6.0) m, 1.79 m
m, 1.60 m (brd, 9.5) s s s s s
1.17 3.37 1.10 1.14 1.00 0.83 0.75
m (dd, 1.5, 10.0) s s s s s
1.15 3.29 1.16 1.22 1.07 1.07 0.74
m, 1.60 m (brd, 10.0) s s s s s
2.06 s 1.17 (t, 7.0) 3.43 (q, 7.0)
Measured at 500 MHz. Measured at 600 MHz.
Fig. 2. Key 1H–1H COSY (bold) and HMBC (arrow) correlations for compounds 1 and 3.
2001) or using modified Mosher’s method (Min et al., 1998). Further confirmation was based on the absolute configuration of 8 which was established by the Cu-Ka X-ray crystallography (Fig. 4). Thus, compound 3 was assigned as (3R,24S)-3-methoxylanost-9(11)-en-24,25-diol. Compound 4 exhibited a [M+Na]+ pseudo molecular ion peak at m/z 481.3672 in the positive HRESIMS, corresponding to the molecular formula of C30H50O3. Comparison of its NMR data to those of 3 showed a general similarity, except that a methoxy group at the C-3 position and a hydroxy moiety at the C-24 position in 3 were replaced by a hydroxy group and a carbonyl unit (dC 215.0), respectively, in 4. By detailed analysis of the HSQC, 1 H–1H COSY, HMBC, and NOESY spectra, compound 4 was then defined as 24-oxolanost-9(11)-en-3R,25-diol. Compound 5 presented a molecular formula C32H54O4 by the positive HRESIMS at m/z 525.3907 [M+Na]+. The NMR data showed overall similarities to those of 8, except that the ketone moiety at the C-3 position in 8 was replaced by an acetyl group [dH 2.06 (3H, s); dC 21.3 CH3, 171.0 C] in 5. The configuration of the acetyl
group at the C-3 position was deduced as b-orientation according to the large coupling constant of H-2 and H-3 (dd, J = 11.5, 4.0 Hz) (Yang et al., 2010b). Thus, compound 5 was elucidated as (3S,24S)-3-acetyl-lanost-9(11)-en-24,25-diol. Compound 6 was assigned the molecular formula C32H56O4 from the positive HRESIMS at m/z 511.4110 [M+Na]+. The 1 H- and 13C-NMR data of 6 were very similar to those of 5. However, close comparison of their 13C-NMR spectra showed a major difference: an acetyl group (dC 21.3 CH3, 171.0 C) in 5 was absent, while an ethoxy group (dC 16.2 CH3, 56.4 CH2) appeared. The HMBC correlation of oxymethylene protons (d 3.43) to C-25 indicated that the ethoxy group was located at the C-25 position. Consequently, compound 6 was established as (3S,24S)-25-ethoxy-lanost-9(11)en-3,24-diol. Since 95% EtOH was used as extraction solvent, compound 6 might be an artifact yielded during the extraction procedure. Compound 7 was assigned the molecular formula C11H16O4 based on the HRESIMS at m/z235.0937. The 1D NMR spectra displayed 11 carbon signals, including a p-hydro-phenyl group
Y.-L. Li et al. / Phytochemistry 106 (2014) 116–123
119
Fig. 3. X-ray crystal structure of compound 1.
Fig. 4. X-ray crystal structure of compound 8.
[dH 6.76, 7.13 (each for 2H, d, J = 8.4 Hz); dC 116.2 d 2, 129.7 d 2, 158.4 s], two oxygenated methine signals [dH 3.64 (1H, dt, J = 3.6, 6.6 Hz), 4.18 (1H, d, J = 6.6 Hz); dC 77.1 d, 83.5 d], one oxygenated methylene [3.27 (1H, dd, J = 6.0, 11.4 Hz), 3.41 (1H, dd, J = 3.6, 11.4 Hz); dC 63.8 d], and an ethoxy group [dH 1.14 (3H, t, J = 6.6 Hz, H-20 ), 3.32 (1H, m, H-10 a), 3.37 (1H, m, H-10 b); dC 15.5 (q, C-20 ), 65.1 (t, C-10 )]. These signals were very similar to those of 1-(4-hydroxyphenyl)-1-methoxyl-2,3-propanediol except that the methoxy moiety was replaced by an ethoxy group in 7. The coupling constant (J = 7.0 Hz) between H-7 and H-8 clearly verified a threo configuration (Yang et al., 2007). Thus, compound 7 was concluded to be (threo)-1-(4-hydroxyphenyl)-1-ethoxy2,3-propanediol. All the isolated compounds were subjected to in vitro bioassay for anti-hepatitis C virus (HCV) infection activity. The anti-HCV
infection activity was evaluated using the n4mBid cell line, which was utilized to report the presence of anti-HCV molecules via increased cell viability relative to untreated cells in the presence of HCV infection. The HCV replication inhibitors 20 -C-methyladenosine was used as the positive control (Chockalingam et al., 2010). Compounds 3, 8, 9, and 10 exhibited significant effects with EC50 values of 3.73, 2.67, 1.33, and 2.25 lM, respectively (Table 4). Furthermore, all isolates were evaluated for the cytotoxic activities against QGY-7703 and A549 tumor cells. However, none of them showed positive effect. The Pinaceae family comprises three sub-families of Abietoideae, Pinoideae, and Laricoideae. Previously, compound 9 was reported from Pinoideae and Laricoideae, Pinus luchuensis (Wada et al., 2001) and Larix kaempferi (Ohtsu et al., 1998). Interestingly, they were reported for the first time from an
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Y.-L. Li et al. / Phytochemistry 106 (2014) 116–123
Table 2 C-NMR data for compounds 1–6 and 8 in CDCl3.
13
No.
1a
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 OCH3
35.6 34.4 217.7 47.2 50.9 20.5 26.3 135.9 148.0 35.8 26.6 32.6 68.1 155.4 27.1 37.8 49.0 18.3 18.5 35.3 16.0 36.3 180.0
2a t t s s d t t s s s t t s s t t s q q d q t s
35.4 34.5 217.4 47.2 50.8 20.4 26.1 135.9 147.4 35.7 26.3 31.5 68.1 155.4 27.0 38.2 49.4 18.4 17.8 34.9 15.2 35.8 79.3 149.6 129.3 174.1 10.5 26.4 21.1 103.7
26.5 q 21.0 q 104.1 t
3b t t s s d t t s s s t t s s t t s q q d q t d d s s q q q t
30.8 21.2 85.9 38.1 47.3 20.4 27.9 41.9 148.6 39.3 114.3 37.1 44.3 47.1 33.9 28.0 50.9 14.4 22.2 36.4 18.5 33.6 28.7 79.6 73.3 23.2 26.5 28.4 22.9 18.5 57.0
4a t t d s d t t d s s d t s s t t d q q d q t t d s q q q q q q
30.5 25.7 76.2 37.9 46.7 21.3 28.0 41.9 148.6 39.4 114.5 37.1 44.3 47.2 33.8 27.9 50.9 14.4 22.1 35.7 18.2 30.3 32.6 215.0 76.2 26.6 26.6 28.3 22.5 18.5
5a t t d s d t t d s s d t s s t t d q q d q t t s s q q q q q
b
t t d s d t t d s s d t s s t t d q q d q t t d s q q q q q
CH3COO
21.3 q
CH3COO
171.0 s
36.2 27.9 79.0 39.2 52.6 21.4 28.0 41.9 148.6 39.4 115.0 37.3 44.4 47.0 34.0 28.2 51.1 14.5 22.3 36.4 18.5 33.9 28.3 77.9 77.4 19.4 21.6 28.3 15.6 18.5
8a t t d s d t t d s s d t s s t t d q q d q t t d s q q q q q
36.7 34.8 217.3 47.7 53.3 22.5 27.6 41.8 147.0 39.0 116.2 37.1 44.2 46.9 33.8 27.9 50.9 14.4 21.8 36.4 18.5 33.5 28.7 79.5 73.2 23.1 26.5 25.6 22.0 18.4
t t s s d t t d s s d t s s t t d q q d q t t d s q q q q q
16.2 q
OCH2CH3 OCH2CH3 a
35.8 24.1 80.8 38.0 52.5 21.2 28.0 41.7 148.1 39.2 115.1 37.1 44.3 47.0 33.8 28.0 50.9 14.4 22.3 36.4 18.5 33.6 28.7 79.6 73.2 23.1 26.5 28.2 16.8 18.5
6a
56.4 t
Measured at 125 MHz. Measured at 150 MHz.
Table 3 1 H- and
Table 4 Anti-hepatitis C virus (HCV) effects of all the isolates from A. nukiangensis. 13
C-NMR data (600 MHz) for compound 7 in CD3OD.
Position
dC 1 2 3 4 5 6 7 8 9 10 20
Compound
7
131.4 s 129.7 d 116.2 d 158.4 s 116.2 d 129.7 d 83.5 d 77.1 d 63.8 t 65.1 t 15.5 q
(3R,24S)-3-Methoxylanost-9(11)-en-24,25-diol (3) 3-Oxolanost-9(11)-en-24S,25-diol (8) Lanost-9(11)-en-3a,24S,25-triol (9) 5,40 -Dihydroxy-3,7-dimethoxy-6-methylflavone (10) 23-Hydroxyl-8(14?13R)-abeo-17,13-friedo-3-oxolanosta8,14(30),24-triene-26,23-olide Other compounds 20 -C-Methyladenosinea
dH 7.13 (d, 8.4) 6.76 (d, 8.4) 6.76 7.13 4.18 3.64 3.41 3.32 1.14
(d, 8.4) (d, 8.4) (d, 6.6) (dt, 3.6, 6.6) (dd, 3.6, 11.4); 3.27 (dd, 6.0, 11.4) m; 3.37 m (t, 6.6)
Abietoideae plant. Thus compound 9 might be of chemotaxonomic significance for the Pinaceae family. From A. nukiangensis, 44 miscellaneous chemical constituents were isolated, including 13 triterpenes, 17 diterpenes, four sesquiterpenes, three lignans, and seven others. Among them, eight were isolated for the first time from Abies species, including three triterpenes: 3-oxolanost9(11)-en-24S,25-diol, lanost-9(11)-en-3a,24S,25-triol, mangiferolic acid; three diterpenes: 13,15-dihydroxypodocarpa-8,11, 13-triene, 7a-hydroxymanool, 8-hydroxy-12-oxo-13-abieten-18oic acid, and two sesquiterpenes: caryolane-1,9b-diol, 8, 9-dihydroxy-1(12)-caryophllene. Compounds 3–6 and 8 are D9,11-lanostanes which were rarely found in Abies species, with few obtained from Abies veitchii (Tanaka and Matsunaga, 1990)
a
EC50 (lM) 3.73 2.67 1.33 2.25 12.01 >100 0.09
Positive control.
and Abies alba (Muller and Ourisson, 1974). Therefore, compounds 3–6 and 8 could have chemotaxonomic significance for A. nukiangensis. 3. Conclusions From A. nukiangensis, seven new and 37 known miscellaneous compounds were obtained. Compound 1 is the first nortriterpene bearing a 8(14?13)abeo-17,13-friedo moiety, and 3–6 are novel D9,11-lanostanes. Compound 9 was found for the first time from the sub-family of Abietoideae, while compounds 3–6 and 8 were rarely found D9,11-lanostanes in Abies species, indicating chemotaxonomic significance of 9 for the Pinaceae family and of 3–6 as well as 8 for A. nukiangensis. In addition, compounds 3 and 8–10 showed significant anti-HCV effects with their EC50 values less than 4 lM.
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4. Experimental 4.1. General experimental procedures Optical rotations were measured with Perkin-Elmer 341 polarimeter. UV and CD spectra were obtained using Shimadzu UV-2550 UV visible and JASCO J810 spectrophotometers, respectively. IR spectra were acquired on a Bruker Vector-22 spectrometer with KBr pellets. NMR spectra were recorded on a Bruker Avance 500 or 600 MHz spectrometer in CDCl3 with TMS as internal standard. EIMS were performed on an Agilent LC/MSD Trap XCT spectrometer, and HRESIMS were completed on an Agilent 6520 AccurateMass Q-TOF LC/MS system. Materials for column chromatography were silica gel (Huiyou Silica Gel Development Co. Ltd., Yantai, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and YMC-GEL ODS-A (YMC, USA). Reversed phase medium pressure liquid chromatography (RP-MPLC) was performed on a Buchi Sepacore system (49 460 mm, v = 20 mL/ min). Preparative TLC was conducted with glass precoated silica gel GF254 (Yantai). 4.2. Plant material The aerial parts of A. nukiangensis were collected in Weixi county, Yunnan Province of China in August 2010, and were identified by Han-Ming Zhang in the school of Pharmacy, Second Military Medical University, China. A herbarium specimen (No. 2010-08-001) was deposited in School of Pharmacy, Second Military Medical University, China. 4.3. Extraction and isolation The powdered aerial parts (5 kg) of A. nukiangensis were extracted with 95% ethanol for 3 3 h. The extracts were combined and concentrated to a small volume. Then it was partitioned with CHCl3 and EtOAc successively. The CHCl3-solute extract was divided into six fractions (C1–C6) by column chromatography (CC) over silica gel eluting with gradient CHCl3–MeOH (100% ? 50%). Fraction C2 was isolated by RP-MPLC eluting with MeOH–H2O (50:50 ? 100:0), followed by CC on Sephadex LH-20 (CHCl3–MeOH, 1:1). Final purification by preparative TLC (CHCl3–MeOH, 30:1 or 50:1) yielded 2 (8 mg), 3 (10 mg), 4 (27 mg), 6 (13 mg), aquilarabietic acid J (11 mg), mangiferonic acid (130 mg), abietic acid (305 mg), abieta-7,13-dien-12a-methoxy18-oic acid (21 mg), dehydroabietic (46 mg), abiesadine P (22 mg), 7-oxodehydroabietinol (5 mg), 9,13b-epidioxy-8 (14)-abieten-18-oic acid (113 mg), 7a-hydroxymanool (6 mg), trans-phytol (9 mg), and 8,9-dihydroxy-1(12)-caryophllene (8 mg). Fraction C3 was separated over Sephadex LH-20 eluting with CHCl3-MeOH (1:1) to give 7 (8 mg), 4-[4-hydroxy-6-(hydroxymethyl)-2,6-dimethyl-1-cyclohex-1-en-1-yl] butan-2-one (8 mg), and dihydrodehydrodiconiferyl alcohol (57 mg). Fraction C4 was separated using flash chromatograph over silica gel eluting with CHCl3–MeOH (95:5), to offer 1 (59 mg), 5 (8 mg), 8 (200 mg), 23-hydroxyl-8(14?13R)-abeo-17,13-friedo-3-oxolanosta-8, 14(30), 24-triene-26,23-olide (30 mg), and isomangiferolic acid (29 mg), mangiferolic acid (160 mg), 18-succinoyloxyabieta-8,11,13-triene (10 mg), 4-hydroxy-3-methoxycinnamaldehyde (20 mg), 4-(phydroxyphenyl)-2-butanone (48 mg), b-sitosterol (150 mg), stigmastanone (13 mg). And fraction C5 was chromatographed on Sephadex LH-20 with MeOH, to give 9 (55 mg), neoabiestrine F (23 mg), 15-hydroxydehydroabietic acid (120 mg), abiesadine N (33 mg), 7a-hydroxydehydroabietic acid (180 mg), 7b-hydroxydehydroabietic acid (17 mg), 13,15-dihydroxypodocarpa-8,11,13triene (2 mg), 8-hydroxy-12-oxo-13-abieten-18-oic acid (8 mg),
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clovane-2b,9a-diol (45 mg), caryolane-1,9b-diol (18 mg), and 10 (157 mg). Fraction C6 was fractionated by RP-MPLC eluting with MeOH–H2O (20:80 ? 100:0), and purified by LH-20 (MeOH) to yield abiesadine I (56 mg), pinoresinol (66 mg), prinsepiol (12 mg), and rhododendrol (26 mg). 4.3.1. 24,25,26,27-Tetranor-8(14?13R)abeo-17,13-friedo-3oxolanosta-8,14(30)-dien-23-oic acid (1) Colorless monoclinic crystals (MeOH); m.p. 140–142 °C; [a]D20: 40.3 (c 0.5, MeOH); IR (KBr) mmax 3440, 2923, 1706, 1646, 1384, 1188, 1112 cm1; 1H- and 13C-NMR data, see Tables 1 and 2; ESIMS (positive) m/z 421[M+Na]+, ESIMS (negative) m/z 397 [MH]; HRESIMS (positive) m/z 399.2900 [M+H]+ (calcd. for C26H39O3, 399.2894). 4.3.2. (13R,23R)-8(14?13)abeo-17,13-friedo-3-Oxolanosta8,14(30),24-trien-26,23-olide (2) Amorphous powder; [a]D20: 15.8 (c 0.30, MeOH); UV (MeOH) kmax (log e) 205 (4.41), 251 (3.61) nm; CD (MeOH) (nm) De212 28.2; IR (KBr) mmax 3448, 2963, 2927, 1754, 1714, 1637, 1384, 1364, 1222 cm1; 1H- and 13C-NMR data, see Tables 1 and 2; ESIMS (positive) m/z 473 [M+Na]+, ESIMS (negative) m/z 485 [M+Cl]; HRESIMS (positive) m/z 451.3208 [M+H]+ (calcd. for C30H43O3, 451.3207). 4.3.3. (3R,24S)-3-Methoxylanost-9(11)-en-24,25-diol (3) Amorphous powder; [a]D20: +23.7 (c 0.39, MeOH); IR (KBr) mmax 3441, 2941, 1634, 1384, 1373, 1102 cm1; 1H- and 13C-NMR data, see Tables 1 and 2; ESIMS (positive) m/z 497 [M+Na]+, ESIMS (negative) m/z 473 [MH]; HRESIMS (positive) [M+Na]+ m/z 497.3973 (calcd. for C31H54O3Na [M+Na]+, 497.3965). 4.3.4. 24-Oxolanost-9(11)-en-3R,25-diol (4) Amorphous powder; [a]D20: +55.0 (c 0.09, MeOH); IR (KBr) mmax 3446, 2980, 2866, 1708, 1470, 1382, 1067, 987 cm1; 1H- and 13 C-NMR data, see Tables 1 and 2; ESIMS (positive) m/z 481 [M+Na]+; ESIMS (negative) m/z 457 [MH]; HRESIMS (positive) m/z 481.3672 [M+Na]+ (calcd. for C30H50O3Na [M+Na]+, 481.3652). 4.3.5. (3S,24S)-3-Acetyl-lanost-9(11)-en-24,25-diol (5) Amorphous powder; [a]D20: +25.5 (c 0.40, MeOH); IR (KBr) mmax 3447, 2923, 1734, 1635, 1384, 1186, 1131 cm1; 1H- and 13C-NMR data, see Tables 1 and 2; ESIMS (positive) m/z 525 [M+Na]+, ESIMS (negative) m/z 501 [MH]; HRESIMS (positive) m/z 525.3907 [M+Na]+ (calcd. for C32H54O4Na, 525.3914). 4.3.6. (3S,24S)-25-Ethoxy-lanost-9(11)-en-3,24-diol (6) Amorphous powder; [a]D20: +54.5 (c 0.05, MeOH); IR (KBr) mmax 3448, 2927, 1636, 1384, 1186 cm1; 1H- and 13C-NMR data, see Tables 1 and 2; ESIMS (positive) m/z 511 [M+Na]+, ESIMS (negative) m/z 487 [MH]; HRESIMS (positive) m/z 511.4110 [M+Na]+ (calcd. for C32H56O3Na, 511.4122). 4.3.7. (threo)-1-(4-Hydroxyphenyl)-1-ethoxy-2,3-propanediol (7) Amorphous powder; [a]D20: 3.0 (c 0.15, MeOH); IR (KBr): mmax 2976, 2925, 1624, 1510, 1372, 1069, 725 cm1; 1H- and 13C-NMR data, see Table 3; ESIMS (positive): m/z 235 [M+Na]+, ESIMS (negative): m/z 211 [MH]; HRESIMS (positive): m/z 235.0937 [M+Na]+ (calcd. for C11H16O4Na: 235.0946). 4.3.8. 3-Oxolanost-9(11)-en-24S,25-diol (8) Colorless orthorhombic crystals (MeOH); 1H- and 13C-NMR data, see Tables 1 and 2; ESIMS (positive) m/z 481 [M+Na]+, ESIMS (negative) m/z 457 [MH], 493 [M+Cl]; HRESIMS (positive) m/z 459.3845 [M+H]+ (calcd. for C30H51O3, 459.3838).
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4.4. X-ray crystallographic data of 24,25,26,27-tetranor-8(14?13R) abeo-17,13- friedo-3-oxolanosta-8,14(30)-dien-23-oic acid (1) Colorless monoclinic crystals of C26H38O3. Formula weight = 398.56. Space group P2(1), a = 7.46140(10) Å, a = 90°; b = 7.80380(10) Å, b = 99.6850(10)°; c = 18.7402(2) Å, c = 90°; V = 1075.64(2) Å3, Z = 2; crystal size 0.20 0.12 0.08 mm3. A total of 8243 unique reflections were collected, of which 3178 were observed (|F|2 P 2r|F|2) using a graphite-monochromator at 140(2) K (Cu-Ka radiation, k = 1.54178 Å) on a Bruker APEX-II CCD diffractometer. 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 = 0.0324, and xR2 = 0.0860 for observed data with I > 2r(I) and flack parameter = 0.03. Crystallographic data have been deposited with the Cambridge Crystallographic Data Center under CCDC 934932. 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]). 4.5. X-ray crystallographic data of 3-oxolanost-9(11)-en-24S,25-diol (8) Colorless orthorhombic crystals of C30H50O3_CH3OH. Formula weight = 490.74. Space group P2(1)2(1)2(1) a = 7.4106(2) Å, a = 90°; b = 11.5382(3) Å, b = 90°; c = 33.6902(9) Å, c = 90°; V = 2880.68(13) Å3, Z = 4; crystal size 0.27 0.16 0.02 mm3. A total of 19,103 unique reflections were collected, of which 5091 were observed (|F|2 P 2r|F|2) using a graphite-monochromator at 140(2) K (Cu-Ka radiation, k = 1.54178 Å) on a Bruker APEX-II CCD diffractometer. 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 = 0.0402 and xR2 = 0.1161 for observed data with I > 2r(I) and flack parameter = 0.14. Crystallographic data have been deposited with the Cambridge Crystallographic Data Center under CCDC 935029. 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]). 4.6. Anti-HCV assay on n4mBid cells According to the reported method (Matsuda et al., 1996), the same volume of n4mBid cells (4.8 105 cells/mL) were mixed with Jc1 HCVcc (3 105–1 106 TCID50/mL). Then 100 lL of the mixture was added to individual wells containing 20 lL of 6 concentrated tested compounds. Plates were maintained at 37 °C in a 5% CO2 atmosphere for 96 h. Cell viability was determined with CellTiter-Glo reagent (Promega) diluted fivefold in water. The stock solutions for all tested compounds (10 mM) were prepared in DMSO. The dose-related study on anti-HCV was carried out to obtain EC50 values. 4.7. Cytotoxic assay As previously reported (Yang et al., 2008b), A549 and QGY-7703 tumor cells were seeded into 96-well plates to incubate at 37 °C with 5% CO2 for 24 h. Then four different concentrations of the tested compounds and the positive controls were added, respectively. And the incubation continued for 48 h. The cell viability was measured using a MTT assay. Acknowledgments The work was supported by program NCET Foundation, NSFC, China (81230090, 21372233, 41176148), Shanghai Leading Aca-
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Chemical constituents of Abies nukiangensis Yong-Li Li a,d, Yan-Xia Gao a, Hui-Zi Jin a, Lei Shan b, Xue-Song Liang e,f, Xi-Ke Xu b, Xian-Wen Yang c,⇑, Ning Wang c, Andre Steinmetz c, Zhilei Chen f, Wei-Dong Zhang a,b,⇑ a
School of Pharmacy, Shanghai JiaoTong University, 800 Dongchuan Road, Shanghai 200240, China School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China c Luxembourg Public Research Centre for Health (CRP-Sante), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg d Division of Material Science & Quality Test, Shanghai Institute of Measurement and Testing Technology, Shanghai 201203, China e Department of Infectious Diseases, Changhai Hospital, Second Military Medical University, Shanghai 200433, China f Artie McFerrin Department of Chemical Engineering, Texas A&M University, TX 77843, USA b
a r t i c l e
i n f o
Article history: Received 9 April 2014 Received in revised form 1 July 2014 Available online 28 July 2014 Keywords: Abies nukiangensis Hepatitis C virus (HCV) Pinaceae Abietoideae Triterpenoids Cu-Ka X-ray crystallography
a b s t r a c t During a survey on chemical constituents of Abies nukiangensis, seven previously unreported compounds, including six triterpenes (1–6) and one phenol (7) were isolated and characterized, together with 37 known miscellaneous chemical constituents. The structures of compounds 1–7 were established mainly by extensive analysis of the 1D and 2D NMR, as well as HRMS data. The absolute configurations of compounds 1 and 8 were confirmed unambiguously by the Cu-Ka X-ray crystallography. Compounds 3 and 8–10 showed significant anti-hepatitis C virus effects with EC50 values of 3.73, 2.67, 1.33 and 2.25 lM, respectively. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Abies plants are a rich source to produce bioactively potent and structurally diverse chemical constituents (Kim et al., 2004; Lavoie et al., 2012; Li et al., 2012b; Wada et al., 2002; Yang et al., 2008a, 2009, 2010b). Since 2006, our research interest has focused on this genus, particularly on those occurring exclusively in Mainland China. Abies nukiangensis Cheng et L.K. Fu (Pinaceae) is a tall tree distributing solely in Mainland China, especially in the northwest areas of Guangxi Province (Zheng and Fu, 1978). As a continuing study, this plant was collected for systematic investigations. Consequently, seven new (1–7, Fig. 1) and 37 known compounds were obtained. Here we report the isolation and structure elucidation of the new compounds. The activities of all the isolates for anti-hepatitis C virus (anti-HCV) on n4mBid cells and cytotoxicity against QGY-7703 and A549 tumor cell lines were evaluated.
The CHCl3-soluble extract of A. nukiangensis was subjected to repeated column chromatography (CC) over silica gel, ODS, and Sephadex LH-20, followed by purification using preparative TLC to yield seven new and 37 known compounds. By comparison of their NMR and MS data with the published references, the known compounds were then identified as seven triterpenes: 3-oxolanost9(11)-en-24S,25-diol (8) (Wada et al., 2001), lanost-9(11)en-3a,24S,25-triol (9) (Ohtsu et al., 1998), neoabiestrine F (Li et al., 2012a), 23-hydroxyl-8(14?13R)-abeo-17,13-friedo-3-oxolanosta-8,14(30),24-triene-26,23-olide (Ou-Yang et al., 2011), isomangiferolic acid (Escobedo-Martínez et al., 2012), mangiferolic acid (Escobedo-Martínez et al., 2012), mangiferonic acid (Escobedo-Martínez et al., 2012); 17 diterpenes: aquilarabietic acid J (Yang et al., 2013), abietic acid (Liu et al., 2009), abieta-7,13dien-12a-methoxy-18-oic acid (Wu et al., 2010), dehydroabietic acid (Cheung et al., 1993), 15-hydroxydehydroabietic acid (Cheung et al., 1993), abiesadine N (Yang et al., 2010a), 7a-hydroxydehydroabietic acid (Cheung et al., 1993), 7b-hydroxydehydroabietic acid (Cheung et al., 1993), 18-succinoyloxyabieta-8,11, 13-triene (Raldugin et al., 2005), abiesadine P (Yang et al., 2010a), 7-oxodehydroabietinol (Tanaka et al., 1997), abiesadine I (Yang et al., 2010a), 9,13b-epidioxy-8(14)-abieten-18-oic acid
⇑ Corresponding authors at: Luxembourg Public Research Center for Health (CRPSante), 84 Val Fleuri, L-1526 Luxembourg, Luxembourg. Tel.: +352 26970 393; fax: +352 26970 390 (X.-W. Yang), School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China. Tel./fax: +86 21 81871244 (W.-D. Zhang). E-mail addresses: [email protected] (X.-W. Yang), wdzhangy@hotmail. com (W.-D. Zhang). http://dx.doi.org/10.1016/j.phytochem.2014.07.003 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Y.-L. Li et al. / Phytochemistry 106 (2014) 116–123
117
Fig. 1. Chemical structures of compounds 1–8.
(Barrero et al., 1991), 13,15-dihydroxypodocarpa-8,11,13-triene (Georges et al., 2012), 7a-hydroxymanool (Jiang et al., 2001), 8-hydroxy-12-oxo-13-abieten-18-oic acid (Baltenweck-Guyot et al., 2000), trans-phytol (Sims et al., 1976); four sesquiterpenes: 4-[4-hydroxy-6-(hydroxymethyl)-2,6-dimethyl-1-cyclohex-1-en1-yl] butan-2-one (Miyase et al., 1987), clovane-2b,9a-diol(Heymann et al., 1994), caryolane-1,9b-diol (Heymann et al., 1994), 8,9-dihydroxy-1(12)-caryophllene (Heymann et al., 1994); three lignans: dihydrodehydrodiconiferyl alcohol (Silva et al., 1989), pinoresinol (Abe and Yamauchi, 1988), prinsepiol (Kilidhar et al., 1982); and six other compounds: 4-hydroxy-3-methoxycinnamaldehyde (Kikuzaki et al., 1999), 5,40 -dihydroxy-3,7-dimethoxy-6methylflavone (10) (Li et al., 2011), rhododendrol (Kubo et al., 1983), 4-(p-hydroxyphenyl)-2-butanone (Ayer and Singer, 1980), b-sitosterol (Hussain et al., 2008), and stigmastanone (Guerriero et al., 1993). Compound 1 was obtained as colorless needle crystals. The HRESIMS of 1 gave a pseudo-molecular ion peak at m/z 399.2900 [M+H]+, consistent with the molecular formula of C26H38O3. The IR spectrum indicated absorption bands of carbonyls (1706 cm1) and olefinic bonds (1646 cm1). The 1H-NMR spectrum of 1 exhibited resonances for four methyl singlets [dH 0.88 (3H, s), 1.07 (6H, s), 1.11 (3H, s)], one methyl doublet [dH 0.94 (3H, d, J = 6.5 Hz)], and one exomethylene [d 4.51 (1H, brs), 4.76 (1H, brs)]. The 13C-NMR spectrum, with the aid of the DEPT technique, resolved 26 carbon signals including five methyls, 10 methylenes (one olefinic), two methines, and nine quaternary carbons (two carbonyls and three olefinic ones) (Table 1). Considering the characteristic signals of one sp3 quaternary carbon (dC 68.1) and two sp2 olefinic bonds (dC 135.9 s, 148.0 s, 104.1 t, 155.4 s), compound 1 was deduced to be a lanostane triterpene containing a 8(14?13R) abeo-17, 13-friedo moiety (Li et al., 2012a; Ou-Yang et al., 2011). The whole planar structure of 1 were deduced by the 1H–1H COSY correlations of five isolated spin systems of H2-1/H2-2, H-5/H2-6/H2-7, H2-11/ H2-12, H2-30/H2-15/H2-16, and H3-21/H-20/H2-22, coupling with the HMBC correlations of Me-18, Me-19, Me-21, Me-28, Me-29, and H2-30 (Fig. 2). Its absolute stereochemistry was established undoubtedly by the Cu-Ka X-ray crystallographic analysis
(Fig. 3). Accordingly, compound 1 was determined unequivocally to be 24,25,26,27-tetranor-8(14?13R)abeo-17,13-friedo-3-oxolanosta-8,14(30)-dien-23-oic acid. Interestingly, compound 1 is the first example of the nortriterpene bearing a 8(14?13) abeo-17,13-friedo moiety. Compound 2 was assigned the molecular formula C30H42O3 from the positive HRESIMS at m/z 451.3208 [M+H]+. The IR spectrum showed the presence of a saturated carbonyl group (1714 cm1) and an a,b-unsaturated butyrolactone (1754 cm1). Its 1H- and 13C-NMR data were in accordance with those of 1 except that the carboxyl moiety at the C-23 position in 1 was substituted by an a,b-unsaturated butyrolactone in 2, which was deduced from the IR absorption (1754 cm1) and the 13C resonances [dC 79.3 (C-23), 149.6 (C-24), 129.3 (C-25), 174.1 (C-26), 10.5 (C-27)]. The assumption was confirmed by the HMBC correlations of H-24 (dH 4.92) to C-22, C-23, C-25, C-26, and C-27. In the CD spectrum, compound 2 exhibited a negative cotton effect at 212 nm (28.2), indicating a R-configuration of the C-23 position (Allen et al., 1971). On the basis of above evidences, 2 was therefore proposed as (13R,23R)-8(14?13)abeo-17,13-friedo-3oxolanosta-8,14(30),24-trien-26,23-olide. Compound 3 was determined to have the molecular formula C31H54O3 from the positive HRESIMS at m/z 497.3973 [M+Na]+. Its NMR spectroscopic data were very similar to those of 3-oxolanost-9(11)-en-24S,25-diol (8), except that the ketone group (dC 217.3) was replaced by an oxygenated methine (dC 85.9). Furthermore, an additional methoxy moiety [dH 3.31(3H, s); dC 57.0] was found, which indicated that a methoxy group was located at the C-3 position in 3 instead of a ketone moiety in 8. The assumption was confirmed by the correlations of H3-28 (dH 0.94), H3-29 (dH 0.88), and the methoxy (dH 3.31) to the oxygenated methine (dC 85.9) in the HMBC spectrum. The a-configuration of C-3 was established according to the small coupling constant of H-3 (dH 2.82, brs) (Yang et al., 2010b), which was further confirmed by the NOESY correlation of H-3 to H3-29 and H3-29 to H3-18. Interestingly, previously isolated lanostane triterpenes containing a 24,25-dihydroxy moiety had all 24S configuration determined either by the CD spectrum using 2% Eu(dpm)3 (Wada et al.,
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Table 1 H-NMR data for compounds 1–6 and 8 in CDCl3 (J in Hz within parenthesis).
1
No.
1a
2a
3b
1 2 3 5 6 7 8 11 12
1.68 m, 1.90 m 2.49 m, 2.60 m
1.64 m, 1.90 m 2.43 m, 2.57 m
1.65 m 1.60 m, 1.69 m 1.95 m, 2.14 m
1.62 m 1.66 m 1.92 m, 2.12 m
1.95 m, 2.14 m 1.44 m, 2.06 m
1.92 m, 2.12 m 1.38 m, 2.12 m
m, 1.81 m m, 2.03 m brs m m, 1.59 m m,1.64 m m (brd, 5.0) m, 2.09 m
1.54 1.76 4.48 0.97 1.48 1.34 2.17 5.23 1.90
m, 1.79 m m (dd, 4.0, 11.5) m m, 1.68 m m,1.67 m (brd, 13.5) (d, 6.0) m, 2.08 m
2.34 m, 2.39 m 1.49 m, 1.56 m
2.33 m, 2.39 m 1.49 m, 1.54 m
0.88 1.07 2.33 0.94 1.99 2.74
0.88 0.92 2.25 0.99 1.38
m, 1.73 m m, 1.60 m brs m m, 1.86 m m,1.67 m m (d, 5.0) m, (brd, 14.0) m m, 1.93 m m s s m (d, 5.5) m, 1.77 m
1.51 1.69 3.44 1.32 1.49 1.34 2.20 5.26 1.90
15 16 17 18 19 20 21 22
1.25 1.39 2.82 1.30 1.72 1.34 2.17 5.23 1.89 2.09 1.34 1.64 1.61 0.65 1.06 1.41 0.90 1.00
1.37 1.64 1.62 0.66 1.07 1.41 0.90 1.27
m m,1.92 m m s s m (d, 7.5) m, 1.82 m
1.35 1.67 1.62 0.65 1.07 1.41 0.90 1.00
1.14 3.29 1.17 1.22 0.94 0.88 0.74 3.31
m, 1.60 m m s s s s s s
2.50 m, 2.56 m
1.13 3.29 1.17 1.22 0.87 0.89 0.74
23 24 26 27 28 29 30 OMe OAc OEt a b
s s m (d, 6.5) (brd, 14.5), (brd, 14.5)
s s m (d, 6.5) m, 1.73 m
4.92 (d, 11.0) 7.01 s
1.11 s 1.07 s 4.51 brs, 4.76 brs
1.87 1.09 1.04 4.46
s s s brs, 4.72 brs
4a
1.39 1.39 0.97 0.89 0.77
5a
s s s s s
6a
8a m, 1.82 m m (dd, 4.0, 12.0) m m, 1.70 m m, 1.68m (brd, 12.0) (d, 6.0) m, (brd, 17.0) m, 1.86 m m m s s m (d, 6.5) m, 1.86 m
1.37 1.63 1.35 2.22 5.28 1.93
m m m, 1.70 m (brd, 12.5) (d, 5.5) m, 2.09 m
m m, 1.91 m m s s m (d, 7.5) m, 1.78 m
1.44 1.75 3.22 0.89 1.50 1.36 2.17 5.23 1.89 2.09 1.37 1.31 1.62 0.66 1.05 1.44 0.90 1.37
1.81 m, 2.10 m 2.40 m, 2.72 m
1.37 1.36 1.62 0.67 1.22 1.42 0.90 1.00
m m, 1.93 m m s s m (d, 6.0) m, 1.79 m
m, 1.60 m (brd, 9.5) s s s s s
1.17 3.37 1.10 1.14 1.00 0.83 0.75
m (dd, 1.5, 10.0) s s s s s
1.15 3.29 1.16 1.22 1.07 1.07 0.74
m, 1.60 m (brd, 10.0) s s s s s
2.06 s 1.17 (t, 7.0) 3.43 (q, 7.0)
Measured at 500 MHz. Measured at 600 MHz.
Fig. 2. Key 1H–1H COSY (bold) and HMBC (arrow) correlations for compounds 1 and 3.
2001) or using modified Mosher’s method (Min et al., 1998). Further confirmation was based on the absolute configuration of 8 which was established by the Cu-Ka X-ray crystallography (Fig. 4). Thus, compound 3 was assigned as (3R,24S)-3-methoxylanost-9(11)-en-24,25-diol. Compound 4 exhibited a [M+Na]+ pseudo molecular ion peak at m/z 481.3672 in the positive HRESIMS, corresponding to the molecular formula of C30H50O3. Comparison of its NMR data to those of 3 showed a general similarity, except that a methoxy group at the C-3 position and a hydroxy moiety at the C-24 position in 3 were replaced by a hydroxy group and a carbonyl unit (dC 215.0), respectively, in 4. By detailed analysis of the HSQC, 1 H–1H COSY, HMBC, and NOESY spectra, compound 4 was then defined as 24-oxolanost-9(11)-en-3R,25-diol. Compound 5 presented a molecular formula C32H54O4 by the positive HRESIMS at m/z 525.3907 [M+Na]+. The NMR data showed overall similarities to those of 8, except that the ketone moiety at the C-3 position in 8 was replaced by an acetyl group [dH 2.06 (3H, s); dC 21.3 CH3, 171.0 C] in 5. The configuration of the acetyl
group at the C-3 position was deduced as b-orientation according to the large coupling constant of H-2 and H-3 (dd, J = 11.5, 4.0 Hz) (Yang et al., 2010b). Thus, compound 5 was elucidated as (3S,24S)-3-acetyl-lanost-9(11)-en-24,25-diol. Compound 6 was assigned the molecular formula C32H56O4 from the positive HRESIMS at m/z 511.4110 [M+Na]+. The 1 H- and 13C-NMR data of 6 were very similar to those of 5. However, close comparison of their 13C-NMR spectra showed a major difference: an acetyl group (dC 21.3 CH3, 171.0 C) in 5 was absent, while an ethoxy group (dC 16.2 CH3, 56.4 CH2) appeared. The HMBC correlation of oxymethylene protons (d 3.43) to C-25 indicated that the ethoxy group was located at the C-25 position. Consequently, compound 6 was established as (3S,24S)-25-ethoxy-lanost-9(11)en-3,24-diol. Since 95% EtOH was used as extraction solvent, compound 6 might be an artifact yielded during the extraction procedure. Compound 7 was assigned the molecular formula C11H16O4 based on the HRESIMS at m/z235.0937. The 1D NMR spectra displayed 11 carbon signals, including a p-hydro-phenyl group
Y.-L. Li et al. / Phytochemistry 106 (2014) 116–123
119
Fig. 3. X-ray crystal structure of compound 1.
Fig. 4. X-ray crystal structure of compound 8.
[dH 6.76, 7.13 (each for 2H, d, J = 8.4 Hz); dC 116.2 d 2, 129.7 d 2, 158.4 s], two oxygenated methine signals [dH 3.64 (1H, dt, J = 3.6, 6.6 Hz), 4.18 (1H, d, J = 6.6 Hz); dC 77.1 d, 83.5 d], one oxygenated methylene [3.27 (1H, dd, J = 6.0, 11.4 Hz), 3.41 (1H, dd, J = 3.6, 11.4 Hz); dC 63.8 d], and an ethoxy group [dH 1.14 (3H, t, J = 6.6 Hz, H-20 ), 3.32 (1H, m, H-10 a), 3.37 (1H, m, H-10 b); dC 15.5 (q, C-20 ), 65.1 (t, C-10 )]. These signals were very similar to those of 1-(4-hydroxyphenyl)-1-methoxyl-2,3-propanediol except that the methoxy moiety was replaced by an ethoxy group in 7. The coupling constant (J = 7.0 Hz) between H-7 and H-8 clearly verified a threo configuration (Yang et al., 2007). Thus, compound 7 was concluded to be (threo)-1-(4-hydroxyphenyl)-1-ethoxy2,3-propanediol. All the isolated compounds were subjected to in vitro bioassay for anti-hepatitis C virus (HCV) infection activity. The anti-HCV
infection activity was evaluated using the n4mBid cell line, which was utilized to report the presence of anti-HCV molecules via increased cell viability relative to untreated cells in the presence of HCV infection. The HCV replication inhibitors 20 -C-methyladenosine was used as the positive control (Chockalingam et al., 2010). Compounds 3, 8, 9, and 10 exhibited significant effects with EC50 values of 3.73, 2.67, 1.33, and 2.25 lM, respectively (Table 4). Furthermore, all isolates were evaluated for the cytotoxic activities against QGY-7703 and A549 tumor cells. However, none of them showed positive effect. The Pinaceae family comprises three sub-families of Abietoideae, Pinoideae, and Laricoideae. Previously, compound 9 was reported from Pinoideae and Laricoideae, Pinus luchuensis (Wada et al., 2001) and Larix kaempferi (Ohtsu et al., 1998). Interestingly, they were reported for the first time from an
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Y.-L. Li et al. / Phytochemistry 106 (2014) 116–123
Table 2 C-NMR data for compounds 1–6 and 8 in CDCl3.
13
No.
1a
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 OCH3
35.6 34.4 217.7 47.2 50.9 20.5 26.3 135.9 148.0 35.8 26.6 32.6 68.1 155.4 27.1 37.8 49.0 18.3 18.5 35.3 16.0 36.3 180.0
2a t t s s d t t s s s t t s s t t s q q d q t s
35.4 34.5 217.4 47.2 50.8 20.4 26.1 135.9 147.4 35.7 26.3 31.5 68.1 155.4 27.0 38.2 49.4 18.4 17.8 34.9 15.2 35.8 79.3 149.6 129.3 174.1 10.5 26.4 21.1 103.7
26.5 q 21.0 q 104.1 t
3b t t s s d t t s s s t t s s t t s q q d q t d d s s q q q t
30.8 21.2 85.9 38.1 47.3 20.4 27.9 41.9 148.6 39.3 114.3 37.1 44.3 47.1 33.9 28.0 50.9 14.4 22.2 36.4 18.5 33.6 28.7 79.6 73.3 23.2 26.5 28.4 22.9 18.5 57.0
4a t t d s d t t d s s d t s s t t d q q d q t t d s q q q q q q
30.5 25.7 76.2 37.9 46.7 21.3 28.0 41.9 148.6 39.4 114.5 37.1 44.3 47.2 33.8 27.9 50.9 14.4 22.1 35.7 18.2 30.3 32.6 215.0 76.2 26.6 26.6 28.3 22.5 18.5
5a t t d s d t t d s s d t s s t t d q q d q t t s s q q q q q
b
t t d s d t t d s s d t s s t t d q q d q t t d s q q q q q
CH3COO
21.3 q
CH3COO
171.0 s
36.2 27.9 79.0 39.2 52.6 21.4 28.0 41.9 148.6 39.4 115.0 37.3 44.4 47.0 34.0 28.2 51.1 14.5 22.3 36.4 18.5 33.9 28.3 77.9 77.4 19.4 21.6 28.3 15.6 18.5
8a t t d s d t t d s s d t s s t t d q q d q t t d s q q q q q
36.7 34.8 217.3 47.7 53.3 22.5 27.6 41.8 147.0 39.0 116.2 37.1 44.2 46.9 33.8 27.9 50.9 14.4 21.8 36.4 18.5 33.5 28.7 79.5 73.2 23.1 26.5 25.6 22.0 18.4
t t s s d t t d s s d t s s t t d q q d q t t d s q q q q q
16.2 q
OCH2CH3 OCH2CH3 a
35.8 24.1 80.8 38.0 52.5 21.2 28.0 41.7 148.1 39.2 115.1 37.1 44.3 47.0 33.8 28.0 50.9 14.4 22.3 36.4 18.5 33.6 28.7 79.6 73.2 23.1 26.5 28.2 16.8 18.5
6a
56.4 t
Measured at 125 MHz. Measured at 150 MHz.
Table 3 1 H- and
Table 4 Anti-hepatitis C virus (HCV) effects of all the isolates from A. nukiangensis. 13
C-NMR data (600 MHz) for compound 7 in CD3OD.
Position
dC 1 2 3 4 5 6 7 8 9 10 20
Compound
7
131.4 s 129.7 d 116.2 d 158.4 s 116.2 d 129.7 d 83.5 d 77.1 d 63.8 t 65.1 t 15.5 q
(3R,24S)-3-Methoxylanost-9(11)-en-24,25-diol (3) 3-Oxolanost-9(11)-en-24S,25-diol (8) Lanost-9(11)-en-3a,24S,25-triol (9) 5,40 -Dihydroxy-3,7-dimethoxy-6-methylflavone (10) 23-Hydroxyl-8(14?13R)-abeo-17,13-friedo-3-oxolanosta8,14(30),24-triene-26,23-olide Other compounds 20 -C-Methyladenosinea
dH 7.13 (d, 8.4) 6.76 (d, 8.4) 6.76 7.13 4.18 3.64 3.41 3.32 1.14
(d, 8.4) (d, 8.4) (d, 6.6) (dt, 3.6, 6.6) (dd, 3.6, 11.4); 3.27 (dd, 6.0, 11.4) m; 3.37 m (t, 6.6)
Abietoideae plant. Thus compound 9 might be of chemotaxonomic significance for the Pinaceae family. From A. nukiangensis, 44 miscellaneous chemical constituents were isolated, including 13 triterpenes, 17 diterpenes, four sesquiterpenes, three lignans, and seven others. Among them, eight were isolated for the first time from Abies species, including three triterpenes: 3-oxolanost9(11)-en-24S,25-diol, lanost-9(11)-en-3a,24S,25-triol, mangiferolic acid; three diterpenes: 13,15-dihydroxypodocarpa-8,11, 13-triene, 7a-hydroxymanool, 8-hydroxy-12-oxo-13-abieten-18oic acid, and two sesquiterpenes: caryolane-1,9b-diol, 8, 9-dihydroxy-1(12)-caryophllene. Compounds 3–6 and 8 are D9,11-lanostanes which were rarely found in Abies species, with few obtained from Abies veitchii (Tanaka and Matsunaga, 1990)
a
EC50 (lM) 3.73 2.67 1.33 2.25 12.01 >100 0.09
Positive control.
and Abies alba (Muller and Ourisson, 1974). Therefore, compounds 3–6 and 8 could have chemotaxonomic significance for A. nukiangensis. 3. Conclusions From A. nukiangensis, seven new and 37 known miscellaneous compounds were obtained. Compound 1 is the first nortriterpene bearing a 8(14?13)abeo-17,13-friedo moiety, and 3–6 are novel D9,11-lanostanes. Compound 9 was found for the first time from the sub-family of Abietoideae, while compounds 3–6 and 8 were rarely found D9,11-lanostanes in Abies species, indicating chemotaxonomic significance of 9 for the Pinaceae family and of 3–6 as well as 8 for A. nukiangensis. In addition, compounds 3 and 8–10 showed significant anti-HCV effects with their EC50 values less than 4 lM.
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4. Experimental 4.1. General experimental procedures Optical rotations were measured with Perkin-Elmer 341 polarimeter. UV and CD spectra were obtained using Shimadzu UV-2550 UV visible and JASCO J810 spectrophotometers, respectively. IR spectra were acquired on a Bruker Vector-22 spectrometer with KBr pellets. NMR spectra were recorded on a Bruker Avance 500 or 600 MHz spectrometer in CDCl3 with TMS as internal standard. EIMS were performed on an Agilent LC/MSD Trap XCT spectrometer, and HRESIMS were completed on an Agilent 6520 AccurateMass Q-TOF LC/MS system. Materials for column chromatography were silica gel (Huiyou Silica Gel Development Co. Ltd., Yantai, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and YMC-GEL ODS-A (YMC, USA). Reversed phase medium pressure liquid chromatography (RP-MPLC) was performed on a Buchi Sepacore system (49 460 mm, v = 20 mL/ min). Preparative TLC was conducted with glass precoated silica gel GF254 (Yantai). 4.2. Plant material The aerial parts of A. nukiangensis were collected in Weixi county, Yunnan Province of China in August 2010, and were identified by Han-Ming Zhang in the school of Pharmacy, Second Military Medical University, China. A herbarium specimen (No. 2010-08-001) was deposited in School of Pharmacy, Second Military Medical University, China. 4.3. Extraction and isolation The powdered aerial parts (5 kg) of A. nukiangensis were extracted with 95% ethanol for 3 3 h. The extracts were combined and concentrated to a small volume. Then it was partitioned with CHCl3 and EtOAc successively. The CHCl3-solute extract was divided into six fractions (C1–C6) by column chromatography (CC) over silica gel eluting with gradient CHCl3–MeOH (100% ? 50%). Fraction C2 was isolated by RP-MPLC eluting with MeOH–H2O (50:50 ? 100:0), followed by CC on Sephadex LH-20 (CHCl3–MeOH, 1:1). Final purification by preparative TLC (CHCl3–MeOH, 30:1 or 50:1) yielded 2 (8 mg), 3 (10 mg), 4 (27 mg), 6 (13 mg), aquilarabietic acid J (11 mg), mangiferonic acid (130 mg), abietic acid (305 mg), abieta-7,13-dien-12a-methoxy18-oic acid (21 mg), dehydroabietic (46 mg), abiesadine P (22 mg), 7-oxodehydroabietinol (5 mg), 9,13b-epidioxy-8 (14)-abieten-18-oic acid (113 mg), 7a-hydroxymanool (6 mg), trans-phytol (9 mg), and 8,9-dihydroxy-1(12)-caryophllene (8 mg). Fraction C3 was separated over Sephadex LH-20 eluting with CHCl3-MeOH (1:1) to give 7 (8 mg), 4-[4-hydroxy-6-(hydroxymethyl)-2,6-dimethyl-1-cyclohex-1-en-1-yl] butan-2-one (8 mg), and dihydrodehydrodiconiferyl alcohol (57 mg). Fraction C4 was separated using flash chromatograph over silica gel eluting with CHCl3–MeOH (95:5), to offer 1 (59 mg), 5 (8 mg), 8 (200 mg), 23-hydroxyl-8(14?13R)-abeo-17,13-friedo-3-oxolanosta-8, 14(30), 24-triene-26,23-olide (30 mg), and isomangiferolic acid (29 mg), mangiferolic acid (160 mg), 18-succinoyloxyabieta-8,11,13-triene (10 mg), 4-hydroxy-3-methoxycinnamaldehyde (20 mg), 4-(phydroxyphenyl)-2-butanone (48 mg), b-sitosterol (150 mg), stigmastanone (13 mg). And fraction C5 was chromatographed on Sephadex LH-20 with MeOH, to give 9 (55 mg), neoabiestrine F (23 mg), 15-hydroxydehydroabietic acid (120 mg), abiesadine N (33 mg), 7a-hydroxydehydroabietic acid (180 mg), 7b-hydroxydehydroabietic acid (17 mg), 13,15-dihydroxypodocarpa-8,11,13triene (2 mg), 8-hydroxy-12-oxo-13-abieten-18-oic acid (8 mg),
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clovane-2b,9a-diol (45 mg), caryolane-1,9b-diol (18 mg), and 10 (157 mg). Fraction C6 was fractionated by RP-MPLC eluting with MeOH–H2O (20:80 ? 100:0), and purified by LH-20 (MeOH) to yield abiesadine I (56 mg), pinoresinol (66 mg), prinsepiol (12 mg), and rhododendrol (26 mg). 4.3.1. 24,25,26,27-Tetranor-8(14?13R)abeo-17,13-friedo-3oxolanosta-8,14(30)-dien-23-oic acid (1) Colorless monoclinic crystals (MeOH); m.p. 140–142 °C; [a]D20: 40.3 (c 0.5, MeOH); IR (KBr) mmax 3440, 2923, 1706, 1646, 1384, 1188, 1112 cm1; 1H- and 13C-NMR data, see Tables 1 and 2; ESIMS (positive) m/z 421[M+Na]+, ESIMS (negative) m/z 397 [MH]; HRESIMS (positive) m/z 399.2900 [M+H]+ (calcd. for C26H39O3, 399.2894). 4.3.2. (13R,23R)-8(14?13)abeo-17,13-friedo-3-Oxolanosta8,14(30),24-trien-26,23-olide (2) Amorphous powder; [a]D20: 15.8 (c 0.30, MeOH); UV (MeOH) kmax (log e) 205 (4.41), 251 (3.61) nm; CD (MeOH) (nm) De212 28.2; IR (KBr) mmax 3448, 2963, 2927, 1754, 1714, 1637, 1384, 1364, 1222 cm1; 1H- and 13C-NMR data, see Tables 1 and 2; ESIMS (positive) m/z 473 [M+Na]+, ESIMS (negative) m/z 485 [M+Cl]; HRESIMS (positive) m/z 451.3208 [M+H]+ (calcd. for C30H43O3, 451.3207). 4.3.3. (3R,24S)-3-Methoxylanost-9(11)-en-24,25-diol (3) Amorphous powder; [a]D20: +23.7 (c 0.39, MeOH); IR (KBr) mmax 3441, 2941, 1634, 1384, 1373, 1102 cm1; 1H- and 13C-NMR data, see Tables 1 and 2; ESIMS (positive) m/z 497 [M+Na]+, ESIMS (negative) m/z 473 [MH]; HRESIMS (positive) [M+Na]+ m/z 497.3973 (calcd. for C31H54O3Na [M+Na]+, 497.3965). 4.3.4. 24-Oxolanost-9(11)-en-3R,25-diol (4) Amorphous powder; [a]D20: +55.0 (c 0.09, MeOH); IR (KBr) mmax 3446, 2980, 2866, 1708, 1470, 1382, 1067, 987 cm1; 1H- and 13 C-NMR data, see Tables 1 and 2; ESIMS (positive) m/z 481 [M+Na]+; ESIMS (negative) m/z 457 [MH]; HRESIMS (positive) m/z 481.3672 [M+Na]+ (calcd. for C30H50O3Na [M+Na]+, 481.3652). 4.3.5. (3S,24S)-3-Acetyl-lanost-9(11)-en-24,25-diol (5) Amorphous powder; [a]D20: +25.5 (c 0.40, MeOH); IR (KBr) mmax 3447, 2923, 1734, 1635, 1384, 1186, 1131 cm1; 1H- and 13C-NMR data, see Tables 1 and 2; ESIMS (positive) m/z 525 [M+Na]+, ESIMS (negative) m/z 501 [MH]; HRESIMS (positive) m/z 525.3907 [M+Na]+ (calcd. for C32H54O4Na, 525.3914). 4.3.6. (3S,24S)-25-Ethoxy-lanost-9(11)-en-3,24-diol (6) Amorphous powder; [a]D20: +54.5 (c 0.05, MeOH); IR (KBr) mmax 3448, 2927, 1636, 1384, 1186 cm1; 1H- and 13C-NMR data, see Tables 1 and 2; ESIMS (positive) m/z 511 [M+Na]+, ESIMS (negative) m/z 487 [MH]; HRESIMS (positive) m/z 511.4110 [M+Na]+ (calcd. for C32H56O3Na, 511.4122). 4.3.7. (threo)-1-(4-Hydroxyphenyl)-1-ethoxy-2,3-propanediol (7) Amorphous powder; [a]D20: 3.0 (c 0.15, MeOH); IR (KBr): mmax 2976, 2925, 1624, 1510, 1372, 1069, 725 cm1; 1H- and 13C-NMR data, see Table 3; ESIMS (positive): m/z 235 [M+Na]+, ESIMS (negative): m/z 211 [MH]; HRESIMS (positive): m/z 235.0937 [M+Na]+ (calcd. for C11H16O4Na: 235.0946). 4.3.8. 3-Oxolanost-9(11)-en-24S,25-diol (8) Colorless orthorhombic crystals (MeOH); 1H- and 13C-NMR data, see Tables 1 and 2; ESIMS (positive) m/z 481 [M+Na]+, ESIMS (negative) m/z 457 [MH], 493 [M+Cl]; HRESIMS (positive) m/z 459.3845 [M+H]+ (calcd. for C30H51O3, 459.3838).
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4.4. X-ray crystallographic data of 24,25,26,27-tetranor-8(14?13R) abeo-17,13- friedo-3-oxolanosta-8,14(30)-dien-23-oic acid (1) Colorless monoclinic crystals of C26H38O3. Formula weight = 398.56. Space group P2(1), a = 7.46140(10) Å, a = 90°; b = 7.80380(10) Å, b = 99.6850(10)°; c = 18.7402(2) Å, c = 90°; V = 1075.64(2) Å3, Z = 2; crystal size 0.20 0.12 0.08 mm3. A total of 8243 unique reflections were collected, of which 3178 were observed (|F|2 P 2r|F|2) using a graphite-monochromator at 140(2) K (Cu-Ka radiation, k = 1.54178 Å) on a Bruker APEX-II CCD diffractometer. 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 = 0.0324, and xR2 = 0.0860 for observed data with I > 2r(I) and flack parameter = 0.03. Crystallographic data have been deposited with the Cambridge Crystallographic Data Center under CCDC 934932. 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]). 4.5. X-ray crystallographic data of 3-oxolanost-9(11)-en-24S,25-diol (8) Colorless orthorhombic crystals of C30H50O3_CH3OH. Formula weight = 490.74. Space group P2(1)2(1)2(1) a = 7.4106(2) Å, a = 90°; b = 11.5382(3) Å, b = 90°; c = 33.6902(9) Å, c = 90°; V = 2880.68(13) Å3, Z = 4; crystal size 0.27 0.16 0.02 mm3. A total of 19,103 unique reflections were collected, of which 5091 were observed (|F|2 P 2r|F|2) using a graphite-monochromator at 140(2) K (Cu-Ka radiation, k = 1.54178 Å) on a Bruker APEX-II CCD diffractometer. 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 = 0.0402 and xR2 = 0.1161 for observed data with I > 2r(I) and flack parameter = 0.14. Crystallographic data have been deposited with the Cambridge Crystallographic Data Center under CCDC 935029. 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]). 4.6. Anti-HCV assay on n4mBid cells According to the reported method (Matsuda et al., 1996), the same volume of n4mBid cells (4.8 105 cells/mL) were mixed with Jc1 HCVcc (3 105–1 106 TCID50/mL). Then 100 lL of the mixture was added to individual wells containing 20 lL of 6 concentrated tested compounds. Plates were maintained at 37 °C in a 5% CO2 atmosphere for 96 h. Cell viability was determined with CellTiter-Glo reagent (Promega) diluted fivefold in water. The stock solutions for all tested compounds (10 mM) were prepared in DMSO. The dose-related study on anti-HCV was carried out to obtain EC50 values. 4.7. Cytotoxic assay As previously reported (Yang et al., 2008b), A549 and QGY-7703 tumor cells were seeded into 96-well plates to incubate at 37 °C with 5% CO2 for 24 h. Then four different concentrations of the tested compounds and the positive controls were added, respectively. And the incubation continued for 48 h. The cell viability was measured using a MTT assay. Acknowledgments The work was supported by program NCET Foundation, NSFC, China (81230090, 21372233, 41176148), Shanghai Leading Aca-
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