Phytochemistry xxx (2015) xxx–xxx
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Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities Guo-Wei Wang a,c, Chao Lv d, Xing Yuan b, Ji Ye b, Hui-Zi Jin a, Lei Shan b, Xi-Ke Xu b, Yun-Heng Shen b,⇑, Wei-Dong Zhang a,b,⇑ a
School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, PR China Department of Phytochemistry, Second Military Medical University, Shanghai 200433, PR China College of Pharmaceutical Sciences and Chinese Medicine, Southwest University, Chongqing 400715, PR China d School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fujian 350108, PR China b c
a r t i c l e
i n f o
Article history: Received 5 October 2014 Received in revised form 22 April 2015 Available online xxxx Keywords: Abies faxoniana Pinaceae Triterpenoids Topoisomerases
a b s t r a c t Nine lanostane-type triterpenoids were isolated from branches and leaves of Abies faxoniana, along with 10 known compounds. Two were isolated as inseparable mixtures of epimers at C-23 of the c-lactone ring that had a lactol structure. The structures of the nine compounds were established by spectroscopic analysis and circular dichroism (CD) data. The absolute configurations at the stereogenic centres of two of the known compounds were confirmed by X-ray crystallography. One compound showed cytotoxic activities against HCT-116, MCF-7, and A549 cells with IC50 values of 8.9, 7.6, and 4.2 lM, respectively. The isolated compounds were tested for their effects on human DNA topoisomerases I and II. One was found to be a selective inhibitor of human topo II activity with an IC50 value of 53.5 lM, which was comparable to that of the topo II inhibitor etoposide (IC50 = 49.6 lM). Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction There are approximately 50 species in the genus Abies (family Pinaceae), 20 of which occur exclusively in China (Fu et al., 1999). Abies plants are a rich source of structurally diverse and biologically important terpenoids (Lavoie et al., 2012; Handa et al., 2013; Huang et al., 1988; Li et al., 2009, 2012; Shan et al., 1988; Yang et al., 2009, 2010a,b). These Abies terpenoids have been demonstrated to exert diverse bioactivities, particularly cytotoxic, antitumor and antiinflammatory activities (Matsunaga et al., 1965; Lavoie et al., 2012; Li et al., 2009, 2012; Wada et al., 2002; Yang et al., 2010a,b). Abies faxoniana is a woody plant distributed exclusively in China, especially in the northern part of the Sichuan province and the southern part of the Gansu province (Fu et al., 1999). In this study, the branches and leaves of A. faxoniana were collected for a systematic chemical investigation, which led to the isolation of 9 new (1–9) and 10 known triterpenoids (Figs. 1 and 2). Herein, the isolation and structural elucidation of compounds 1–9, their cytotoxic activities against human ⇑ Corresponding authors at: Department of Phytochemistry, Second Military Medical University, Shanghai 200433, PR China (W.-D. Zhang and Y.-H. Shen). Tel.: +86 21 81871244. E-mail addresses: [email protected] (Y.-H. Shen), wdzhangy@hotmail. com (W.-D. Zhang).
tumour cell lines, and their inhibitory effects on human DNA topoisomerases I and II are described.
2. Results and discussion The CH2Cl2 soluble part of the EtOH extract from the branches and leaves of A. faxoniana was fractionated by silica gel column chromatography, followed by Sephadex LH-20 and preparative HPLC to afford 9 new lanostane-type triterpenoids (1–9) and 10 known compounds (10–19). The known compounds were determined to be abieslactone (Matsunaga et al., 1965; Kutney and Westcott, 1971) (10), 3b-hydroxy-9b-lanosta-7,24-dien-26,23 R-olide (Tanaka and Matsunaga, 1991) (11), 3-oxo-9bH-lanost7-en-26,23-olide (Kukina et al., 1998) (12), (23R,25R)-3,4seco-17,14-friedo-9bH-lanosta-4(28),6,8(14)-trien-26,23-olid-3-oic acid (Wada et al., 2002) (13), (23R,25R)-3,4-seco-9bH-lanosta4(28),7-dien-26,23-olid-3-oic acid (Raldugin et al., 1986) (14), 7,14,22Z,24-mariesatetraen-26,23-olide-3a-ol (Gao et al., 2008) (15), abiesatrine D (Yang et al., 2010b) (16), firmanoic acid (Hasegawa et al., 1987a) (17), neoabieslactone E (Li et al., 2009) (18), and spiromarienonol A (Tanaka et al., 2004) (19) by comparing their spectroscopic data with those reported in the literature. The absolute configurations at the stereogenic centres of known compounds 10 and 16 were confirmed by X-ray crystallography
http://dx.doi.org/10.1016/j.phytochem.2015.04.012 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang, G.-W., et al. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.012
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G.-W. Wang et al. / Phytochemistry xxx (2015) xxx–xxx
Fig. 1. Chemical structures of new compounds 1–9.
(the single crystal X-ray diffraction data are found in Fig. S70 in the Supplementary data). Compound 1 was assigned the molecular formula C30H44O3 from its positive HRESIMS with a [M+H]+ ion at a m/z of 453.3302, indicating a hydrogen deficiency index of nine. Its IR spectrum indicated absorption bands for a,b-unsaturated-c-lactone (1741 cm 1), carbonyl (1698 cm 1), and olefinic (1646 cm 1) groups. Its 1H and 13C NMR spectra showed a 4-substituted 2methyl-2-butenolide ring [dH 1.91 (t), 4.98 (ddd), 6.99 (d); dC 10.6 (q), 78.9 (d), 129.4 (s), 149.7 (d), 174.4 (s)], a tetrasubstituted double bond [dC 135.1 (s), 133.2 (s)], a carbonyl group [dC 217.8 (s)], five methyl groups attached to tertiary carbons, a secondary methyl group, and nine methylene groups. The above spectroscopic data of 1 including those of the lactone moiety resembled those of the known compound 3-oxo-9b-lanosta-7,24-dien26,23R-olide (Tanaka et al., 1990), except for the presence of an olefinic group at C-8 and C-9 instead of at C-7 and C-8. Thus, structure 1 was assigned as 3-oxolanosta-8,24-dien-23,26-olide. In the NOESY spectrum, H-17 correlated with H3-30, suggesting a 17R⁄ relative configuration of 1. In the circular dichroism (CD) spectrum of 1 (Fig. S9 in the Supplementary data), a negative Cotton effect at 216 nm ( 12.1) was found, which established a 23R configuration of the lactone side-chain in 1 (Allen et al., 1971; Tanaka et al., 2004; Li et al., 2009). Accordingly, the structure of compound 1 was proposed to be 3-oxolanosta-8,24-dien-26,23R-olide, and it was named neoabieslactone G. Neoabieslactone H (2) shared the same molecular formula as 1, and they exhibited similar IR and NMR spectroscopic data. However, comparison of the 1H and 13C NMR spectra of the two compounds showed a difference in the double bond position. HMBC correlations from H3-19 to C-9 and from H-11 to C-9 and C-12 indicated that the double bond in 2 was located at the C-9 and C-11 positions [dH 5.28 (dd); dC 147.1 (s), 116.0 (d)], instead of the double bond at the C-8 and C-9 positions of 1. The 17R⁄
relative configuration of 2 was established on the basis of the NOESY correlation of H-17 with H3-30. The 23R-configuration of the lactone side-chain in 2 was deduced from CD measurements (Fig. S18 in the Supplementary data), which gave rise to a negative Cotton effect at 216 nm ( 9.4) similar to that of 1. Therefore, compound 2 was deduced to be 3-oxolanosta-9(11),24-dien-26,23Rolide. Compound 3 had a molecular formula of C30H42O3 as determined by positive HRESIMS with a [M+H]+ ion at a m/z of 451.3127, indicating a hydrogen deficiency index of ten. The IR spectrum indicated absorption bands for a,b-unsaturated-c-lactone (1745 cm 1), carbonyl (1696 cm 1), and olefinic (1646 and 1648 cm 1) groups. The 1H and 13C NMR spectroscopic data of 3 showed overall similarities to those of the known compound 3oxo-9b-lanosta-7,24-dien-26,23R-olide (Tanaka et al., 1990), except for the presence of an additional double bond at C-22 and C-23 [dH 5.44 (d); dC 147.1 (s), 120.1 (d)]. In the NOESY spectrum, H-17 was correlated with H3-30, indicating that C-17 has a R⁄ configuration in 3. Compound 3 was thus assigned as 3-oxo-9blanosta-7,22E,24-trien-26,23-olide, and given the name neoabieslactone I. The molecular formula of compound 4 was established to be C30H48O3 on the basis of its [M H] ion at a m/z of 455.3610 in negative HRESIMS, indicating a hydrogen deficiency index of seven. The IR spectrum suggested the presence of hydroxy (3445 cm 1), carboxylic (1772 cm 1), and olefinic (1646 cm 1) groups. The NMR spectroscopic data were very similar to those of the known compound abiesatrine D (Yang et al., 2010b), except for the presence of a C-3 hydroxy group [dC 77.5 (d)] in 4 instead of a carbonyl moiety [dC 219.3 (s)]. The hydroxy group in 4 could be located at C-3 because of the HMBC correlations from H3-28 and H3-29 to the oxymethine at dC 77.5. In the NOESY spectrum (Fig. 3), H-3 was correlated to H3-19, which established a 3a-OH group in 4. The 17R⁄ relative configuration of 4 was established
Please cite this article in press as: Wang, G.-W., et al. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.012
G.-W. Wang et al. / Phytochemistry xxx (2015) xxx–xxx
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Fig. 2. Chemical structures of compounds 10–19.
Fig. 3. Key 1H–1H COSY, HMBC, and NOESY correlations for 4.
Fig. 4. Key 1H–1H COSY, HMBC, and NOESY correlations for 5.
on the basis of the NOESY correlation of H-17 with H3-30. Consequently, the structure of compound 4 was determined to be 3a-hydroxy-9b-lanosta-7,24E-dien-26-oic acid, and it was named abiesatrine K.
Compound 5 was determined to have the molecular formula C30H46O4 from the [M+H]+ ion at a m/z of 471.3383 in positive HRESIMS, indicating a hydrogen deficiency index of eight. A comparison of its 1D NMR spectroscopic data with those of firmanoic
Please cite this article in press as: Wang, G.-W., et al. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.012
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G.-W. Wang et al. / Phytochemistry xxx (2015) xxx–xxx
Fig. 5. The structures of (S)- and (R)-PGME amides of 5 and their Dd values.
acid showed a general similarity (Hasegawa et al., 1987a), except that the olefinic carbons located at the C-24 and C-25 positions of firmanoic acid were absent, while olefinic carbons appeared at C-14 and C-15 for 5 [dH 5.18 (dd); dC 152.5 (s), 115.2 (d)]. Moreover, the ketone group at C-3 in firmanoic acid was substituted by a hydroxyl group [dH 3.22 (dd); dC 79.5 (d)] in 5. In the NOESY spectrum, H3-18 correlated with H-20, suggesting a 17S⁄ relative configuration of 5. The relative configuration of the C-3 hydroxy moiety was deduced to have a b-orientation according to the NOESY correlation of H-3 with H3-28 and the large coupling constant of H-2 and H-3 (dd, J = 12.0, 4.0 Hz) (Yang et al., 2010b; Li et al., 2014) (Fig. 4). The absolute stereochemistry at C-25 was
Fig. 6. Key 1H–1H COSY, HMBC, and NOESY correlations for 6.
Table 1 H (500 MHz) NMR spectroscopic data for compounds 1–9 in CDCl3.
1
No.
1
1
1.63 1.82 2.39 2.58
2
2 m m m m
1.60 1.82 2.24 2.71
3 m m m m
1.62 1.81 2.38 2.50
4 m m m m
3 5 6 7 8 9 11
1.60 dd (14.0, 3.5) 1.90 m 2.10 m 2.05 m
1.61 dd (14.0, 3.5) 1.80 m 2.08 m 2.10 m
5.28 dd (6.1, 3.0) 1.88 m 2.40 m 1.35 m 1.70 m 1.82 m 2.07 m 1.52 m 0.69 s 1.58 s 1.71 m 1.02 d (6.4) 1.35 m 1.53 m 4.98 ddd (11.0,2.5,1.5) 6.99 d (1.5)
25 27
1.91 s
28 29 30
1.06 s 0.87 s 1.08 s
15 16 17 18 19 20 21 22 23 24
m m m m brs
1.41 dd (14.0, 3.5) 1.82 m 1.91 m 5.63 dd (6.5, 3.0)
1.45 dd (14.0, 3.5) 1.85 m 1.93 m 5.51 dd (6.5, 3.0)
2.23 m 1.38 m 1.65 m 1.64 m 1.85 m 1.37 m 1.78 m 1.80 m 1.97 m 1.68 m 0.82 s 0.97 s 2.51 m 1.02 d (6.4) 5.44 d (11.0)
2.18 m 1.40 m 1.63 m 1.32 m 1.80 m 1.40 m 1.78 m 1.80 m 1.96 m 1.47 m 0.96 s 0.89 s 1.61 m 0.85 d (6.5) 1.51 m
5
6
1.54 m 1.82 m 1.57 m 1.96 m 3.22 dd (4.0, 12.0) 1.60 dd (14.0, 3.5) 1.83 m 1.92 m 5.56 dd (6.5, 3.0)
1.54 1.83 1.58 1.95 3.30
7
1.45 dd (14.0, 3.5) 1.83 m 1.92 m 5.65 dd (6.5, 3.0)
1.45 dd (14.0, 3.5) 1.83 m 1.92 m 5.67 dd (6.5, 3.0)
1.41 dd (14.0, 3.5) 1.82 m 1.91 m 5.52 dd (6.5, 3.0)
1.54 m 1.84 m 1.59 m 1.95 m 3.44 dd (4.0, 12.0) 1.45 dd (14.0, 3.5) 1.85 m 1.93 m 5.54 dd (6.5, 3.0)
2.23 1.38 1.63 1.50 1.86 5.18 (1.2, 1.90 2.20
2.23m 1.81 m 2.25 m 5.54 dd (6.5, 3.0) 1.40 m 1.73 m 1.44 m 1.92 m
2.23 1.38 1.63 1.51 1.86 1.62 1.84 5.28 (6.5,
2.23 1.81 2.25 5.53 (6.5, 1.40 1.73 1.46 1.92
2.23 1.81 2.25 5.53 (6.5, 1.40 1.74 1.44 1.91
0.95 s 0.94 s 2.21 m 0.88 d (6.5) 2.23 m 2.90 m
1.08 s 1.03 s 2.10 m 1.07 d (6.5) 2.33 m 2.82 m
1.13 s 0.89 s 1.60 m 0.97 d (6.5) 1.91 m
1.14 s 1.08 s 1.61 m 0.97 d (6.5) 1.91m
7.15 s
6.64 s
6.66 s
6.77 s 2.18 s
2.17 s
2.14 s
2.15 s
0.93 s 0.92 s 1.20 s
0.93 s 0.85 s 1.09 s
1.06 s 0.93 s 1.20 s
0.94 s 0.90 s 1.23 s
m m m m brs
1.60 1.83 2.36 2.58
8 m m m m
1.66 1.81 2.38 2.50
9 m m m m
1.91 m 1.35 m 1.62 m 1.50 m 2.07 m 1.35 m 1.73 m 1.82 m 1.96 m 1.50 m 0.73 s 1.11 s 1.73 m 1.02 d (6.4) 1.37 m 1.51 m 4.98 ddd (11.0,2.5,1.5) 6.99 d (1.5)
12
1.54 1.84 1.59 1.95 3.34
m m m m m dd 2.8) m m
0.87 s 1.00 s 2.42 m 0.80 d (6.5) 2.30 m 2.47 m
m m m m m m m dd 3.0)
m m m dd 3.0) m m m m
m m m dd 3.0) m m m m
2.02 m 7.30 s
6.61 m
1.91 s
2.00 s
2.48 s
1.23 s 1.08 s 0.73 s
1.08 s 1.08 s 1.02 s
0.83 s 0.73 s 1.70 s
2.57 m 2.82 m 2.83 m 1.19 d (7.1) 1.01 s 0.92 s 0.86 s
Please cite this article in press as: Wang, G.-W., et al. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.012
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G.-W. Wang et al. / Phytochemistry xxx (2015) xxx–xxx Table 2 C (125 MHz) NMR spectroscopic data for compounds 1–9 in CDCl3.
13
No.
1
2
3
4
5
6
7
8
9
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
36.0 t 34.6 t 217.8 s 47.4 s 51.2 d 19.3 t 26.2 t 135.1 s 133.2 s 36.8 s 21.0 t 30.7 t 44.6 s 49.9 s 30.9 t 28.0 t 50.8 d 15.9 q 18.6 q 33.7 d 18.6 q 40.6 t 78.9 d 149.7 d 129.4 s 174.4 s 10.6 q 26.1 q 21.2 q 24.2 q
36.7 t 34.8 t 217.1 s 47.6 s 53.4 d 27.6 t 22.4 t 41.7 d 147.1 s 39.0 s 116.0 d 37.1 t 44.4 s 47.0 s 33.7 t 27.9 t 51.3 d 14.5 q 22.0 q 33.3 d 18.3 q 40.6 t 78.9 d 149.6 d 129.4 s 174.4 s 10.6 q 25.5 q 21.7 q 18.3 q
34.2 t 34.0 t 219.1 s 46.9 s 52.4 d 22.9 t 121.8 d 148.1 s 45.2 d 35.8 s 20.5 t 33.0 t 44.2 s 52.0 s 33.9 t 28.6 t 52.7 d 22.5 q 23.1 q 35.1 d 20.6 q 120.1 d 147.1 s 134.0 d 130.0 s 171.1 s 10.7 q 28.0 q 21.1 q 27.1 q
35.5 t 28.1 t 77.5 d 38.8 s 48.5 d 23.1 t 121.6 d 148.8 s 48.4 d 35.8 s 22.8 t 33.4 t 43.5 s 52.7 s 34.7 t 28.5 t 53.3 s 24.7 q 17.1 q 35.9 d 18.4 q 35.0 t 25.5 t 142.4 d 127.8 s 169.3 s 12.6 q 29.5 q 23.7 q 30.8 q
34.8 t 27.2 t 79.5 d 38.6 s 43.3 d 23.1 t 120.5 d 136.4 s 52.7 d 34.9 s 33.4 t 25.3 t 51.5 s 152.5 s 115.2 d 44.9 t 50.4 s 28.3 q 16.4 q 34.0 d 16.8 q 46.3 t 208.9 s 46.6 t 34.5 d 180.8 s 15.7 q 22.3 q 19.2 q 16.9 q
29.2 t 25.1 t 75.8 d 36.5 s 37.9 d 22.9 t 122.3 d 146.0 s 51.2 d 34.5 s 27.7 t 118.6 d 155.9 s 49.7 s 36.5 t 37.7 t 46.1 s 25.2 q 15.0 q 38.7 d 21.4 q 48.0 t 204.3 s 150.4 s 126.6 d 175.0 s 14.7 q 24.0 q 22.2 q 27.5 q
34.0 t 33.4 t 219.8 s 46.8 s 53.7 d 22.6 t 121.3 d 146.5 s 43.9 d 36.2 s 18.1 t 25.6 t 52.0 s 50.4 s 39.6 t 120.6 d 156.0 s 26.7 q 23.0 q 28.6 d 20.6 q 51.5 t 201.1 s 134.2 d 139.2 s 171.9 s 13.9 q 25.1 q 21.3 q 28.3 q
36.6 t 29.2 t 211.2 s 46.6 s 38.1 d 23.1 t 122.7 d 145.7 s 50.5 d 34.7 s 25.2 t 118.5 d 156.1 s 50.0 s 36.6 t 38.3 t 36.9 s 25.2 q 14.0 q 37.8 d 17.8 q 40.1 t 107.3 s 147.7 d 131.5 s 172.4 s 10.3 q 28.1 q 22.0 q 22.9 q
36.7 t 29.2 t 76.7 d 46.6 s 38.1 d 23.1 t 122.8 d 145.7 s 50.5 d 34.7 s 25.2 t 118.6 d 156.2 s 50.1 s 36.7 t 38.3 t 36.9 s 25.2 q 17.8 q 37.8 d 22.0 q 40.2 t 107.2 s 147.6 d 131.5 s 172.3 s 10.3 q 28.1 q 22.0 q 22.9 q
determined by the PGME (phenylglycine methyl ester) method (Nagai and Kusumi, 1995; Handa et al., 2013). The (S)- and (R)PGME amides were obtained after 5 was treated with (R)- and (S)-PGME, respectively (Fig. 5). Thus, the absolute configuration of 25R for 5 could be elucidated according to the Dd values (Dd = dS dR) (Fig. 5). As such, compound 5 was defined as 3b-hydroxy-23-oxomariesia-7,14-dien-26-oic acid, and named abiesatrine L. Compound 6 was shown to have the molecular formula C30H44O4, as determined by the [M+H]+ ion peak at a m/z of 469.3237 in the positive HRESIMS, indicating a hydrogen deficiency index of nine. It had almost the same IR, 1H and 13C NMR spectroscopic data as those of the known compound 23-oxo-mariesiic acid B (Hasegawa et al., 1987b), except that CH3-27 was attached to C-24 due to the observation of the HMBC correlation of the methyl H3-27 [dH 2.18 (s)] with C-23 [dC 204.3 (s)], C-24 [dC 150.4 (s)] and C-25 [dC 126.6 (d)] (Fig. 6). In the NOESY spectrum, H-20 correlated with H3-30, indicating that C-17 in 6 had a S⁄ configuration. The a-orientation of the hydroxyl group at C-3 was established based on the NOESY correlation of H-3 with H3-19. Compound 6 was thus identified as 3a-hydroxy-23oxomariesia-7,12,24E-trien-26-oic acid and named abiesatrine M.
Compound 7 was assigned a molecular formula C30H42O4 from the [M H] ion peak at a m/z 465.3077 in negative HRESIMS, indicating a hydrogen deficiency index of ten. Compound 7 also showed similar 1H and 13C NMR spectra to those of the known compound 23-oxo-mariesiic acid B (Hasegawa et al., 1987b), except for the presence of a ketone moiety [dC 219.8 (s)] at the C-3 position of 7, instead of the 3-OH of 23-oxo-mariesiic acid B. This feature was verified by the HMBC correlations from H3-28 and H3-29 to the carbonyl carbon. The structure of compound 7 was then concluded to be 3,23-dioxomariesia-7,16,24E-trien26-oic acid, and given the name abiesatrine N. Neoabieslactone J (8) was deduced to have the molecular formula C30H42O4 from the [M+H]+ ion at a m/z of 467.3101 in positive HRESIMS. The 1H and 13C NMR spectra were quite similar to those of 23-hydroxy-3-oxo-9b-lanosta-7,24-dien-26,23-olide (Raldugin et al., 1989), except for the presence of an additional olefinic bond at C-12 and C-13 [dH 5.53 (dd); dC 118.5 (d), 156.1 (s)]. In the HMBC spectrum, H3-18 showed a long-range correlation with the olefinic carbon at dC 156.1 (C-13), and H-12 [dH 5.53 (dd)] was correlated with C-11 and C-13. These correlations suggested that the C-12 and C-13 positions of 8 accounted for a double bond. In the 1H and 13C NMR spectra of 8, typical resonance signals of the
Fig. 7. Key 1H–1H COSY, HMBC, and NOESY correlations for 9.
Please cite this article in press as: Wang, G.-W., et al. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.012
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Table 3 The inhibition of the isolated compounds from A. faxoniana on DNA topo II activity. Compounds
IC50 (lM)
Compounds
IC50 (lM)
Compounds
IC50 (lM)
1 2 3 4 5 6 7
>200 >200 53.5 >200 74.1 72.3 101.2
8 9 10 11 12 13 14
>200 >200 >200 >200 >200 >200 >200
15 16 17 18 19 Etoposide
>200 >200 >200 >200 >200 49.6
side-chain, including one hemiketal carbon at dC 107.3 (C-23), two olefinic carbon at dC 147.7 and 131.5 (C-24 and C-25), one ester carbonyl carbon at dC 172.4 (C-26), and one singlet methyl proton at dH 2.14 (s, H3-27), indicated that 8 has a c-lactone ring with a lactol as in neoabieslactone E (Li et al., 2009), 7,14,24-mariesatrien-26,23-olide-3a,23-diol (Gao et al., 2008), and 3a,23dihydroxylanosta-9(11),16,24-trien-26,23-olide (Handa et al., 2013). In the HMBC spectrum of 8, an olefinic proton at dH 6.64 (s, H-24) exhibited long-range correlations with C-23, C-26, and C-27. In the NOESY spectrum of 8, a methyl proton (CH3-27) was correlated with the olefinic proton (H-24). A methyl doublet (dH 0.97, 3H, J = 6.5 Hz, H-21) and its HMBC-correlated carbon signals [dC 36.9 (C-17), 37.8 (C-20), 40.1 (C-22)] were assigned to the side-chain moiety with a lactone ring. In the 1H and 13C NMR spectra of 8, some resonances showed broad weak peaks or minor peaks, pretty close to those of a type of previously reported lanostane triterpenoid featuring a c-lactone moiety with a lactol (Li et al., 2009; Handa et al., 2013). Gao et al. noted that these types of triterpenoids are usually tautomeric mixtures due to the C-23 epimerisation of the c-lactone moiety with a hemiketal (Gao et al., 2008). Therefore, the main resonance signals in the 1H and 13 C NMR spectra of 8 (Tables 1 and 2) were attributed to this phenomenon and were used to determine the structure of 8. On the basis of the above evidence, the structure of compound 8 was assigned as 23-hydroxy-3-oxo-9b-lanosta-7,12,24-trien-26, 23-olide. The molecular formula of C30H44O4 for compound 9 was determined from the [M H] ion at a m/z of 467.3236 in negative HRESIMS. The 1H and 13C NMR spectra of 9 showed similar broad weak peaks or minor peaks to those of 8, indicating that 9 possessed the same c-lactone moiety with a lactol as 8 and was also a tautomeric mixture due to C-23 epimerisation. Consequently, the structure of 9 was elucidated by the main resonance signals in the NMR spectrum. Analysis of the 1H and 13C NMR spectra exhibited that the NMR data of 9 was quite close to those of 8, except for the presence of a hydroxy group [dH 3.44 (t); dC 76.7 (d)] at C-3 in 9 instead of a ketone moiety as in 8. In the NOESY spectrum, H-3 was correlated with H-5 and H3-28, suggesting that the relative configuration of 3-OH was a b-configuration (Fig. 7). Consequently, the structure of compound 9 was elucidated to be 3b,23-dihydroxy-9b-lanosta-7,12,24-trien-26,23-olide, and it was named neoabieslactone K.
The isolated compounds were tested for their in vitro cytotoxic activities against the human tumour cell lines HepG2, Huh7, SMMC7721, HCT-116, MCF-7, and A549. Compound 3 demonstrated cytotoxicities against HCT-116, MCF-7, and A549 cells with IC50 values of 8.9, 7.6, and 4.2 lM, respectively. Meanwhile, compound 5 had IC50 values of 17.9, 20.1, and 25.3 lM for the human hepatoma cell lines HepG2, Huh7, and SMMC7721, respectively. Furthermore, the isolates were evaluated for their inhibitory effects on human DNA topos I and II in vitro. The conversion of supercoiled pHOT-1 DNA to relaxed DNA by topos I and II was examined in the presence of these compounds. None of these compounds inhibited DNA relaxation by topo I, even when the concentrations were increased up to 200 lM. However, compounds 3, 5, 6, and 7 showed complete inhibition of topo II relaxation activity at 200 lM. Compounds 8 and 9 showed 18% and 9% inhibition, respectively, against topo II activity at 200 lM. Compounds 3, 5, and 6 had inhibitory effects against topo II with IC50 values of 53.5, 74.1, and 72.3 lM, respectively, which were comparable to that of the topo II inhibitor etoposide (IC50 = 49.6 lM) (Table 3). 3. Conclusions From the branches and leaves of A. faxoniana, 9 new (1–9) and 10 known triterpenoids were isolated. Compounds 1–3 have a a,b-unsaturated-c-lactone ring in the C-17 side-chain. Compounds 8 and 9 were obtained as epimeric mixtures potentially arising from tautomerism at C-23 of the c-lactone structure. A plausible biogenetic pathway of the lactone structure in compounds 1–3, and 8–9 is shown in Fig. 8. These compounds might originate from the corresponding 23-keto type lanostanes. Their biosynthesis might provide some insight into the structural transformation of lanostane-type triterpenoids. Since triterpenoids from some Abies plants displayed cytotoxic and antitumor effects, the isolated compounds were tested for their cytotoxic activities and DNA topoisomerase inhibitory effects. Compound 3 showed cytotoxic activities against HCT-116, MCF-7, and A549 cell lines. It also had inhibitory effect against topo II with IC50 value of 53.5 lM. Further evaluation of the inhibition mechanism by compound 3 is now in progress. 4. Experimental 4.1. General experimental procedures 1D and 2D NMR spectrum were determined with a Bruker Avance-500 spectrometer. ESIMS were acquired on an Agilent LC/MSD Trap XCT micromass spectrometer, whereas HRESIMS were measured using an Agilent 6520 Accurate-Mass Q-TOF LC/MS. Optical rotations were obtained with a JASCO P-2000 polarimeter. UV spectrum were obtained on a Shimadzu UV2550 spectrometer. IR spectrum were recorded on a Bruker FTIR Vector 22 spectrometer using KBr pellets. Column chromatography
Fig. 8. Postulated biosynthetic pathway of the lactone structure in the side-chain.
Please cite this article in press as: Wang, G.-W., et al. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.012
G.-W. Wang et al. / Phytochemistry xxx (2015) xxx–xxx
(CC) was performed on silica gel (100–200, 200–300 mesh, Shandong, China), Sephadex LH-20 (GE Healthcare, Sweden) and YMC 50 lm ODS-A (Milford, MA, USA), respectively. Preparative TLC (0.4–0.5 mm, 20 20 cm) was conducted with glass plates precoated with silica gel GF254 (Yantai, Shandong, China). A semi-preparative column (Agilent ZORBAX SB-C18, 5 lm, 9.4 250 mm) and analytical column (Agilent ZORBAX ExtendC18, 5 lm, 4.6 250 mm) were used for HPLC (Shimadzu LC-2010A HT). 4.2. Plant material The branches and leaves of A. faxoniana were collected from Li county, Sichuan province, in August 2009 and authenticated by Prof. Han-Ming Zhang in the Department of Pharmacognosy, Second Military Medical University. A voucher specimen (20090813001) was deposited at the Herbarium of the School of Pharmacy, Second Military Medical University, Shanghai, People’s Republic of China. 4.3. Extraction and isolation Air-dried branches and leaves of A. faxoniana (5.3 kg) were powdered and extracted with EtOH:H2O (80:20, v/v) three times under conditions of reflux. The solvent was removed in vacuo to afford a crude EtOH extract, which was suspended in H2O and then successively partitioned with petroleum ether (PE), CH2Cl2, EtOAc, and nBuOH. The CH2Cl2 extract (100.0 g) was subjected to silica gel (/ 8 100 cm; 100–200 mesh, 1000 g) CC and eluted with a PE/EtOAc gradient (50:1–1:1) to give 10 fractions (Fr.1–Fr.10) based on TLC analysis. Fr.5 [PE/EtOAc (10:1), 30.0 g] underwent CC (/ 4.5 60 cm) on silica gel (200–300 mesh, 700 g) and was eluted with a gradient of PE/EtOAc (20:1–1:1) to afford 8 subfractions (Fr.5-1–Fr.5-8). Subfraction Fr.5-3 [PE/EtOAc (10:1), 4.0 g] was subjected to semi-preparative HPLC (MeOH/H2O, 50:50) to give 7 (40.0 mg), 18 (20.0 mg), and 19 (30.0 mg). Fr.6 [PE/EtOAc (8:1), 28.0 g] was applied to a silica gel column (/ 4.5 60 cm; 200–300 mesh, 600 g) and was eluted with a PE/EtOAc gradient (10:1–1:1) to give 8 subfractions (Fr.6-1–Fr.6-8). From subfraction Fr.6-5 [PE/EtOAc (5:1), 3.0 g], compounds 1 (50.0 mg), 9 (25.0 mg), 13 (50.0 mg), and 14 (28.0 mg) were isolated after CC over Sephadex LH-20 (/ 4.0 150 cm; MeOH) followed by semipreparative HPLC (MeOH/H2O, 50:50). Fr.6–7 [PE/EtOAc (3:1), 3.6 g] was subjected to CC on silica gel (/ 2.5 30 cm; 50 g, 200–300 mesh) and was eluted with a PE/EtOAc gradient (10:1– 1:1) followed by semi-preparative HPLC (MeOH/H2O, 50:50) to give 2 (12.0 mg), 5 (24.6 mg), and 6 (15.4 mg). Compound 10 (40.0 mg) was crystallised from Fr.6–6 [PE/EtOAc (4:1), 1.6 g]. Compounds 11 (30.0 mg) and 12 (25.0 mg) were crystallised from Fr.6-8 [PE/EtOAc (1:1), 2.0 g]. Fr.6-4 [PE/EtOAc (6:1), 2.2 g] was subjected to CC on silica gel (/ 2.5 30 cm; 50 g, 200–300 mesh) and was eluted with a CH2Cl2/MeOH gradient (10:1–1:1) to give 5 subfractions (Fr.6-4a–Fr.6-4e). Fr.6–4e [CH2Cl2/MeOH (1:1), 0.6 g] was subjected to semi-preparative HPLC (MeOH/H2O, 50:50) to give 3 (10.0 mg), 15 (16.0 mg), and 16 (15.0 mg) and was then further eluted with (MeOH/H2O, 60:40) to yield 4 (50.0 mg), 8 (18.0 mg), and 17 (20.0 mg). 4.3.1. Neoabieslactone G (1) Amorphous powder; [a]20 D +51 (c 0.1, CHCl3); UV (MeOH) kmax (log e) 206.9 (3.10) nm; IR (KBr) mmax 3070, 3022, 2930, 2852, 1741, 1698, 1646, 1460, 1410, 1378, 1360, 1197, 1095, 1040, 933, 878, 818 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (positive) m/z 453.3302 [M+H]+ (calcd for C30H45O3, 453.3290).
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4.3.2. Neoabieslactone H (2) Amorphous powder; [a]20 D +29 (c 0.1, CHCl3); UV (MeOH) kmax (log e) 208.0 (3.90) nm; IR (KBr) mmax 3069, 3020, 2928, 2852, 1740, 1698, 1648, 1460, 1410, 1378, 1360, 1197, 1095, 1040, 933, 878, 818 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (positive) m/z 453.3296 [M+H]+ (calcd for C30H45O3, 453.3290). 4.3.3. Neoabieslactone I (3) Amorphous powder; [a]20 34 (c 0.1, CHCl3); UV (MeOH) kmax D (log e) 278.0 (3.60) nm; IR (KBr) mmax 3069, 3020, 2928, 2852, 1745, 1696, 1648, 1646, 1460, 1410, 1378, 1360, 1197, 1095, 1040, 933, 878, 818 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (positive) m/z 451.3127 [M+H]+ (calcd for C30H43O3, 451.3134). 4.3.4. Abiesatrine K (4) Amorphous powder; [a]20 D +53 (c 0.1, CHCl3); UV (MeOH) kmax (log e) 205.0 (2.80) nm; IR (KBr) mmax 3445, 2925, 1772, 1698, 1646, 1465, 1378, 1045, 818 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (negative) m/z 455.3610 [M H] (calcd for C30H47O3, 455.3603). 4.3.5. Abiesatrine L (5) Amorphous powder; [a]20 D +54 (c 0.1, CHCl3); UV (MeOH) kmax (log e) 230.0 (3.80) nm; IR (KBr) mmax 3345, 2928, 1769, 1698, 1646, 1460, 1410, 1378, 1360, 1040, 933, 878 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (positive) m/z 471.3383 [M+H]+ (calcd for C30H47O4, 471.3396). 4.3.6. Abiesatrine M (6) Amorphous powder; [a]20 100 (c 0.1, CHCl3); UV (MeOH) kmax D (log e) 296.0 (4.12) nm; IR (KBr) mmax 3345, 2928, 1769, 1698, 1648, 1646, 1460, 1410, 1378, 1360, 1040, 878 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (positive) m/z 469.3237 [M+H]+ (calcd for C30H45O4, 469.3240). 4.3.7. Abiesatrine N (7) Amorphous powder; [a]20 D +113 (c 0.1, CHCl3); UV (MeOH) kmax (log e) 236.1 (4.01) nm; IR (KBr) mmax 2928, 1769, 1698, 1648, 1646, 1460, 1410, 1378, 1360, 1040, 878 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (negative) m/z 465.3077 [M H] (calcd for C30H41O4, 465.3083). 4.3.8. Neoabieslactone J (8) Amorphous powder; [a]20 D +16 (c 0.1, CHCl3); UV (MeOH) kmax (log e) 210.0 (4.71) nm; IR (KBr) mmax 3419, 2917, 1754, 1700, 1437, 1207, 954, 705 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (positive) m/z 467.3101 [M+H]+ (calcd for C30H43O4, 467.3083). 4.3.9. Neoabieslactone K (9) Amorphous powder; [a]20 12 (c 0.1, CHCl3); UV (MeOH) kmax D (log e) 212.1 (4.65) nm; IR (KBr) mmax 3453, 2995, 2914, 1755, 1463, 1311, 1054, 954, 700 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (negative) m/z 467.3236 [M H] (calcd for C30H43O4, 467.3240). 4.3.10. (S)- and (R)-PGME amides of 5 To 5 (2.0 mg) in N,N-dimethylformamide (DMF, 0.5 mL) was added (S)-PGME (5.0 mg). Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop, 10.0 mg), 1-hydroxybenzotriazole (HOBT, 4.0 mg), and N-methylmorpholine (30 lL) were then successively added. The reaction mixture was moved to a refrigerator at 4 °C overnight. The mixture was then stirred for 2 h at room temperature and subjected to HPLC
Please cite this article in press as: Wang, G.-W., et al. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.012
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(Agilent ZORBAX SB-C18, 5 lm, 9.4 250 mm; solvents CH3CN– 0.2%CF3CO2H, 90:10; detector, UV 210 nm), yielding the (S)-PGME amide of 5 (0.8 mg). The same procedure was used to prepare the (R)-PGME amide of 5 (0.8 mg). 4.3.11. (S)-PGME amide of 5 Amorphous powder; ESI-MS m/z 618 [M+H]+, 1H NMR (500 MHz, CDCl3) dH 5.53 (1H, dd, J = 6.5, 3.0 Hz, H-7), 5.14 (1H, dd, J = 1.2, 2.8 Hz, H-15), 0.80 (3H, s, H-18), 1.00 (3H, s, H-19), 2.35 (1H, m, H-20), 0.73 (3H, d, J = 6.5 Hz, H-21), 2.22 (1H, m, H22), 2.37 (1H, dd, J = 14.1, 1.8 Hz, H-22), 2.53 (1H, m, H-24), 2.83 (1H, m, H-24), 2.83 (1H, m, H-25), 1.29 (3H, d, J = 7.1 Hz, H-27), 1.01 (3H, s, H-28), 0.92 (3H, s, H-29), 0.78 (3H, s, H-30). 4.3.12. (R)-PGME amide of 5 Amorphous powder; ESI-MS m/z 618 [M+H]+, 1H NMR (500 MHz, CDCl3) dH 5.56 (1H, dd, J = 6.5, 3.0 Hz, H-7), 5.18 (1H, dd, J = 1.2, 2.8 Hz, H-15), 0.87 (3H, s, H-18), 1.00 (3H, s, H-19), 2.42 (1H, m, H-20), 0.80 (3H, d, J = 6.5 Hz, H-21), 2.30 (1H, m, H22), 2.47 (1H, dd, J = 14.1, 1.8 Hz, H-22), 2.57 (1H, m, H-24), 2.82 (1H, m, H-24), 2.83 (1H, m, H-25), 1.19 (3H, d, J = 7.1 Hz, H-27), 1.01 (3H, s, H-28), 0.92 (3H, s, H-29), 0.86 (3H, s, H-30).
4.6. DNA topoisomerase I inhibitory activity assay Reactions were carried out in the same manner as described for the topo II relaxation assays, except that the reaction mixtures contained 10 mM Tris–HCl (pH = 7.9), 1 mM EDTA, 150 mM NaCl, 0.1% BSA, 0.1 mM spermidine, 5% glycerol, and supercoiled pHOT-1 DNA (0.25 lg). Acknowledgments We thank Ms. Yangyun Zhou of Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, PR China, for assisting with DNA topoisomerase inhibitory activities. The work was supported by program NCET Foundation, National Natural Science Foundation of China (NSFC, 81230090), Shanghai Leading Academic Discipline Project (B906), Key laboratory of drug research for special environments, PLA, Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300), the Scientific Foundation of Shanghai China (12401900801, 13401900101), National Major Project of China (2011ZX09307-002-03) and the National Key Technology R&D Program of China (2012BAI29B06). Appendix A. Supplementary data
4.4. Cytotoxic assay Cell viability was determined by a MTT assay (Yang et al., 2008b). Briefly, cells were seeded in 96-well plates at 6 103 cells/well and exposed to the test compounds (0, 1, 5, 10, 25, and 50 lM) for 24 h. The cultures were treated with DMSO as the vehicle control. After 24 h of treatment, MTT solution 10 lL (5 mg/mL; Sigma; St. Louis, MO) was added to each well, and the plates were incubated for 2–4 h at 37 °C. The supernatant was then removed from the formazan crystals, and DMSO 100 lL was added to each well. The absorbance at 570 nm was measured using an OPTImax microplate reader. The cell viability was calculated by dividing the mean optical density (OD) of compound-containing wells by that of the DMSO-control wells. 4.5. DNA topoisomerase II inhibitory activity assay Supercoiled pHOT-1 DNA, 5X universal stop buffer, human DNA topos I (recombinant) and IIa (p170 form) were purchased from TopoGen, Inc. (Columbus, OH). The DNA topo inhibitors etoposide and camptothecin were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). The topo II activity was measured by assessing the relaxation of supercoiled pHOT-1 DNA (Muller et al., 1988; Wada et al., 2001). The reaction mixture contained 50 mM Tris–HCl (pH = 8.0), 150 mM NaCl, 10 mM MgCl2, 5 mM ATP, 0.5 mM dithiothreitol, 30 lg bovine serum albumin (BSA)/mL, supercoiled pHOT-1 DNA (0.2 lg), the indicated drug concentrations (1% DMSO), and 1 U of topo IIa in a final volume of 20 lL. The reaction mixtures were incubated for 30 min at 37 °C and were then terminated with 5 stop buffer (5 lL per 20 lL reaction volume). The stop buffer contained 5% sarkosyl, 0.0025% bromophenol blue, and 25% glycerol. The reaction products were electrophoresed on a 1% agarose gel in TAE (tris–acetate–EDTA) running buffer at 8 V/cm for 1 h. The gels were stained with ethidium bromide (0.5 lg/mL) for 1 h and destained in water for 30 min. For the quantitative analysis of the topo II activity, the gels were directly scanned with a FLA2000 fluoroimage analyser, and the area representing supercoiled DNA was calculated. The IC50 values were determined by interpolation from plots of the topo II activity versus the inhibitor concentration.
Supplementary data associated with this article, including the CD spectrum for compounds 1 and 2, the HRESIMS, 1D and 2D NMR spectrum for compounds 1–9 as well as X-ray crystallographic structures of compounds 10 and 16 can be found, in the online version. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.phytochem.2015.04.012. References Allen, F.H., Kutney, J.P., Trotter, J., Westcott, N.D., 1971. The structures and absolute stereochemistry of cyclograndisolide and epicyclograndisolide, novel triterpene lactones from Abies grandis. Tetrahedron Lett. 12, 283–286. Fu, L.G., Li, N., Thomas, S.E., 1999. Flora of China, vol. 4. Science Press, Beijing, pp. 11–52. Gao, H.Y., Wu, L.J., Nakane, T., Shirota, O., Kuroyanagi, M., 2008. Novel lanostane and rearranged lanostane-type triterpenoids from Abies sachalinensis. Chem. Pharm. Bull. 56, 554–558. Handa, M., Murata, T., Kobayashi, K., Selenge, E., Miyase, T., Batkhuu, J., Yoshizaki, F., 2013. Lipase inhibitory and LDL anti-oxidative triterpenes from Abies sibirica. Phytochemistry 86, 168–175. Hasegawa, S., Kaneko, N., Hirose, Y., 1987a. Triterpenes from the seed of Abies firma. Phytochemistry 26, 1095–1099. Hasegawa, S., Miura, T., Kaneko, N., Hirose, Y., Iitaka, Y., 1987b. Further new rearranged lanostanoids from the seeds of Abies mariesii and A. firma. Tetrahedron 43, 1775–1784. Huang, Y., Wen, M., Xiao, S., Zhao, H., Ren, W., 1988. The chemical components of essential oil from Abies faxoniana. Acta Bot. Yunnan 10, 109–112. Kukina, T.P., Shakirov, M.M., Raldugin, V.A., 1998. Triterpenoids from Abies species. 23. A lanostane analog of cytotoxic cycloartanolide and other lactones from needle-free shoots of Siberian fir. Russ. Chem. Bull. 47, 2009–2011. Kutney, J.P., Westcott, N.D., 1971. Structure of abieslactone. Tetrahedron Lett. 37, 3463–3466. Lavoie, S., Legault, J., Gauthier, C., Mshvildadze, V., Mercier, S., Pichette, A., 2012. Abibalsamins A and B, two new tetraterpenoids from Abies balsamea oleoresin. Org. Lett. 14, 1504–1507. Li, Y.L., Yang, X.W., Li, S.M., Shen, Y.H., Zeng, H.W., Liu, X.H., Tang, J., Zhang, W.D., 2009. Terpenoid constituents of Abies chensiensis with potential antiinflammatory activity. J. Nat. Prod. 72, 1065–1068. Li, Y.L., Gao, Y.X., Yang, X.W., Jin, H.Z., Ye, J., Simmons, L., Wang, N., Steinmetz, A., Zhang, W.D., 2012. Cytotoxic triterpenoids from Abies recurvata. Phytochemistry 81, 159–164. Li, Y.L., Gao, Y.X., Jin, H.Z., Shan, L., Liang, X.S., Xu, X.K., Yang, X.Y., Wang, N., Steinmetz, A., Chen, Z., Zhang, W.D., 2014. Chemical constituents of Abies nukiangensis. Phytochemistry 106, 116–123. Matsunaga, S., Okada, J., Uyeo, S., 1965. The structure of abieslactone, a methoxytetracyclic triterpene lactone. Chem. Commun. 21, 525–527. Muller, M.T., Spitzner, J.R., DiDonato, J.A., Mehta, V.B., Tsutsui, K., Tsutsui, K., 1988. Single-strand DNA cleavages by eukaryotic topoisomerase II. Biochemistry 27, 8369–8379.
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Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities Guo-Wei Wang a,c, Chao Lv d, Xing Yuan b, Ji Ye b, Hui-Zi Jin a, Lei Shan b, Xi-Ke Xu b, Yun-Heng Shen b,⇑, Wei-Dong Zhang a,b,⇑ a
School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, PR China Department of Phytochemistry, Second Military Medical University, Shanghai 200433, PR China College of Pharmaceutical Sciences and Chinese Medicine, Southwest University, Chongqing 400715, PR China d School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fujian 350108, PR China b c
a r t i c l e
i n f o
Article history: Received 5 October 2014 Received in revised form 22 April 2015 Available online xxxx Keywords: Abies faxoniana Pinaceae Triterpenoids Topoisomerases
a b s t r a c t Nine lanostane-type triterpenoids were isolated from branches and leaves of Abies faxoniana, along with 10 known compounds. Two were isolated as inseparable mixtures of epimers at C-23 of the c-lactone ring that had a lactol structure. The structures of the nine compounds were established by spectroscopic analysis and circular dichroism (CD) data. The absolute configurations at the stereogenic centres of two of the known compounds were confirmed by X-ray crystallography. One compound showed cytotoxic activities against HCT-116, MCF-7, and A549 cells with IC50 values of 8.9, 7.6, and 4.2 lM, respectively. The isolated compounds were tested for their effects on human DNA topoisomerases I and II. One was found to be a selective inhibitor of human topo II activity with an IC50 value of 53.5 lM, which was comparable to that of the topo II inhibitor etoposide (IC50 = 49.6 lM). Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction There are approximately 50 species in the genus Abies (family Pinaceae), 20 of which occur exclusively in China (Fu et al., 1999). Abies plants are a rich source of structurally diverse and biologically important terpenoids (Lavoie et al., 2012; Handa et al., 2013; Huang et al., 1988; Li et al., 2009, 2012; Shan et al., 1988; Yang et al., 2009, 2010a,b). These Abies terpenoids have been demonstrated to exert diverse bioactivities, particularly cytotoxic, antitumor and antiinflammatory activities (Matsunaga et al., 1965; Lavoie et al., 2012; Li et al., 2009, 2012; Wada et al., 2002; Yang et al., 2010a,b). Abies faxoniana is a woody plant distributed exclusively in China, especially in the northern part of the Sichuan province and the southern part of the Gansu province (Fu et al., 1999). In this study, the branches and leaves of A. faxoniana were collected for a systematic chemical investigation, which led to the isolation of 9 new (1–9) and 10 known triterpenoids (Figs. 1 and 2). Herein, the isolation and structural elucidation of compounds 1–9, their cytotoxic activities against human ⇑ Corresponding authors at: Department of Phytochemistry, Second Military Medical University, Shanghai 200433, PR China (W.-D. Zhang and Y.-H. Shen). Tel.: +86 21 81871244. E-mail addresses: [email protected] (Y.-H. Shen), wdzhangy@hotmail. com (W.-D. Zhang).
tumour cell lines, and their inhibitory effects on human DNA topoisomerases I and II are described.
2. Results and discussion The CH2Cl2 soluble part of the EtOH extract from the branches and leaves of A. faxoniana was fractionated by silica gel column chromatography, followed by Sephadex LH-20 and preparative HPLC to afford 9 new lanostane-type triterpenoids (1–9) and 10 known compounds (10–19). The known compounds were determined to be abieslactone (Matsunaga et al., 1965; Kutney and Westcott, 1971) (10), 3b-hydroxy-9b-lanosta-7,24-dien-26,23 R-olide (Tanaka and Matsunaga, 1991) (11), 3-oxo-9bH-lanost7-en-26,23-olide (Kukina et al., 1998) (12), (23R,25R)-3,4seco-17,14-friedo-9bH-lanosta-4(28),6,8(14)-trien-26,23-olid-3-oic acid (Wada et al., 2002) (13), (23R,25R)-3,4-seco-9bH-lanosta4(28),7-dien-26,23-olid-3-oic acid (Raldugin et al., 1986) (14), 7,14,22Z,24-mariesatetraen-26,23-olide-3a-ol (Gao et al., 2008) (15), abiesatrine D (Yang et al., 2010b) (16), firmanoic acid (Hasegawa et al., 1987a) (17), neoabieslactone E (Li et al., 2009) (18), and spiromarienonol A (Tanaka et al., 2004) (19) by comparing their spectroscopic data with those reported in the literature. The absolute configurations at the stereogenic centres of known compounds 10 and 16 were confirmed by X-ray crystallography
http://dx.doi.org/10.1016/j.phytochem.2015.04.012 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang, G.-W., et al. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.012
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Fig. 1. Chemical structures of new compounds 1–9.
(the single crystal X-ray diffraction data are found in Fig. S70 in the Supplementary data). Compound 1 was assigned the molecular formula C30H44O3 from its positive HRESIMS with a [M+H]+ ion at a m/z of 453.3302, indicating a hydrogen deficiency index of nine. Its IR spectrum indicated absorption bands for a,b-unsaturated-c-lactone (1741 cm 1), carbonyl (1698 cm 1), and olefinic (1646 cm 1) groups. Its 1H and 13C NMR spectra showed a 4-substituted 2methyl-2-butenolide ring [dH 1.91 (t), 4.98 (ddd), 6.99 (d); dC 10.6 (q), 78.9 (d), 129.4 (s), 149.7 (d), 174.4 (s)], a tetrasubstituted double bond [dC 135.1 (s), 133.2 (s)], a carbonyl group [dC 217.8 (s)], five methyl groups attached to tertiary carbons, a secondary methyl group, and nine methylene groups. The above spectroscopic data of 1 including those of the lactone moiety resembled those of the known compound 3-oxo-9b-lanosta-7,24-dien26,23R-olide (Tanaka et al., 1990), except for the presence of an olefinic group at C-8 and C-9 instead of at C-7 and C-8. Thus, structure 1 was assigned as 3-oxolanosta-8,24-dien-23,26-olide. In the NOESY spectrum, H-17 correlated with H3-30, suggesting a 17R⁄ relative configuration of 1. In the circular dichroism (CD) spectrum of 1 (Fig. S9 in the Supplementary data), a negative Cotton effect at 216 nm ( 12.1) was found, which established a 23R configuration of the lactone side-chain in 1 (Allen et al., 1971; Tanaka et al., 2004; Li et al., 2009). Accordingly, the structure of compound 1 was proposed to be 3-oxolanosta-8,24-dien-26,23R-olide, and it was named neoabieslactone G. Neoabieslactone H (2) shared the same molecular formula as 1, and they exhibited similar IR and NMR spectroscopic data. However, comparison of the 1H and 13C NMR spectra of the two compounds showed a difference in the double bond position. HMBC correlations from H3-19 to C-9 and from H-11 to C-9 and C-12 indicated that the double bond in 2 was located at the C-9 and C-11 positions [dH 5.28 (dd); dC 147.1 (s), 116.0 (d)], instead of the double bond at the C-8 and C-9 positions of 1. The 17R⁄
relative configuration of 2 was established on the basis of the NOESY correlation of H-17 with H3-30. The 23R-configuration of the lactone side-chain in 2 was deduced from CD measurements (Fig. S18 in the Supplementary data), which gave rise to a negative Cotton effect at 216 nm ( 9.4) similar to that of 1. Therefore, compound 2 was deduced to be 3-oxolanosta-9(11),24-dien-26,23Rolide. Compound 3 had a molecular formula of C30H42O3 as determined by positive HRESIMS with a [M+H]+ ion at a m/z of 451.3127, indicating a hydrogen deficiency index of ten. The IR spectrum indicated absorption bands for a,b-unsaturated-c-lactone (1745 cm 1), carbonyl (1696 cm 1), and olefinic (1646 and 1648 cm 1) groups. The 1H and 13C NMR spectroscopic data of 3 showed overall similarities to those of the known compound 3oxo-9b-lanosta-7,24-dien-26,23R-olide (Tanaka et al., 1990), except for the presence of an additional double bond at C-22 and C-23 [dH 5.44 (d); dC 147.1 (s), 120.1 (d)]. In the NOESY spectrum, H-17 was correlated with H3-30, indicating that C-17 has a R⁄ configuration in 3. Compound 3 was thus assigned as 3-oxo-9blanosta-7,22E,24-trien-26,23-olide, and given the name neoabieslactone I. The molecular formula of compound 4 was established to be C30H48O3 on the basis of its [M H] ion at a m/z of 455.3610 in negative HRESIMS, indicating a hydrogen deficiency index of seven. The IR spectrum suggested the presence of hydroxy (3445 cm 1), carboxylic (1772 cm 1), and olefinic (1646 cm 1) groups. The NMR spectroscopic data were very similar to those of the known compound abiesatrine D (Yang et al., 2010b), except for the presence of a C-3 hydroxy group [dC 77.5 (d)] in 4 instead of a carbonyl moiety [dC 219.3 (s)]. The hydroxy group in 4 could be located at C-3 because of the HMBC correlations from H3-28 and H3-29 to the oxymethine at dC 77.5. In the NOESY spectrum (Fig. 3), H-3 was correlated to H3-19, which established a 3a-OH group in 4. The 17R⁄ relative configuration of 4 was established
Please cite this article in press as: Wang, G.-W., et al. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.012
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Fig. 2. Chemical structures of compounds 10–19.
Fig. 3. Key 1H–1H COSY, HMBC, and NOESY correlations for 4.
Fig. 4. Key 1H–1H COSY, HMBC, and NOESY correlations for 5.
on the basis of the NOESY correlation of H-17 with H3-30. Consequently, the structure of compound 4 was determined to be 3a-hydroxy-9b-lanosta-7,24E-dien-26-oic acid, and it was named abiesatrine K.
Compound 5 was determined to have the molecular formula C30H46O4 from the [M+H]+ ion at a m/z of 471.3383 in positive HRESIMS, indicating a hydrogen deficiency index of eight. A comparison of its 1D NMR spectroscopic data with those of firmanoic
Please cite this article in press as: Wang, G.-W., et al. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.012
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G.-W. Wang et al. / Phytochemistry xxx (2015) xxx–xxx
Fig. 5. The structures of (S)- and (R)-PGME amides of 5 and their Dd values.
acid showed a general similarity (Hasegawa et al., 1987a), except that the olefinic carbons located at the C-24 and C-25 positions of firmanoic acid were absent, while olefinic carbons appeared at C-14 and C-15 for 5 [dH 5.18 (dd); dC 152.5 (s), 115.2 (d)]. Moreover, the ketone group at C-3 in firmanoic acid was substituted by a hydroxyl group [dH 3.22 (dd); dC 79.5 (d)] in 5. In the NOESY spectrum, H3-18 correlated with H-20, suggesting a 17S⁄ relative configuration of 5. The relative configuration of the C-3 hydroxy moiety was deduced to have a b-orientation according to the NOESY correlation of H-3 with H3-28 and the large coupling constant of H-2 and H-3 (dd, J = 12.0, 4.0 Hz) (Yang et al., 2010b; Li et al., 2014) (Fig. 4). The absolute stereochemistry at C-25 was
Fig. 6. Key 1H–1H COSY, HMBC, and NOESY correlations for 6.
Table 1 H (500 MHz) NMR spectroscopic data for compounds 1–9 in CDCl3.
1
No.
1
1
1.63 1.82 2.39 2.58
2
2 m m m m
1.60 1.82 2.24 2.71
3 m m m m
1.62 1.81 2.38 2.50
4 m m m m
3 5 6 7 8 9 11
1.60 dd (14.0, 3.5) 1.90 m 2.10 m 2.05 m
1.61 dd (14.0, 3.5) 1.80 m 2.08 m 2.10 m
5.28 dd (6.1, 3.0) 1.88 m 2.40 m 1.35 m 1.70 m 1.82 m 2.07 m 1.52 m 0.69 s 1.58 s 1.71 m 1.02 d (6.4) 1.35 m 1.53 m 4.98 ddd (11.0,2.5,1.5) 6.99 d (1.5)
25 27
1.91 s
28 29 30
1.06 s 0.87 s 1.08 s
15 16 17 18 19 20 21 22 23 24
m m m m brs
1.41 dd (14.0, 3.5) 1.82 m 1.91 m 5.63 dd (6.5, 3.0)
1.45 dd (14.0, 3.5) 1.85 m 1.93 m 5.51 dd (6.5, 3.0)
2.23 m 1.38 m 1.65 m 1.64 m 1.85 m 1.37 m 1.78 m 1.80 m 1.97 m 1.68 m 0.82 s 0.97 s 2.51 m 1.02 d (6.4) 5.44 d (11.0)
2.18 m 1.40 m 1.63 m 1.32 m 1.80 m 1.40 m 1.78 m 1.80 m 1.96 m 1.47 m 0.96 s 0.89 s 1.61 m 0.85 d (6.5) 1.51 m
5
6
1.54 m 1.82 m 1.57 m 1.96 m 3.22 dd (4.0, 12.0) 1.60 dd (14.0, 3.5) 1.83 m 1.92 m 5.56 dd (6.5, 3.0)
1.54 1.83 1.58 1.95 3.30
7
1.45 dd (14.0, 3.5) 1.83 m 1.92 m 5.65 dd (6.5, 3.0)
1.45 dd (14.0, 3.5) 1.83 m 1.92 m 5.67 dd (6.5, 3.0)
1.41 dd (14.0, 3.5) 1.82 m 1.91 m 5.52 dd (6.5, 3.0)
1.54 m 1.84 m 1.59 m 1.95 m 3.44 dd (4.0, 12.0) 1.45 dd (14.0, 3.5) 1.85 m 1.93 m 5.54 dd (6.5, 3.0)
2.23 1.38 1.63 1.50 1.86 5.18 (1.2, 1.90 2.20
2.23m 1.81 m 2.25 m 5.54 dd (6.5, 3.0) 1.40 m 1.73 m 1.44 m 1.92 m
2.23 1.38 1.63 1.51 1.86 1.62 1.84 5.28 (6.5,
2.23 1.81 2.25 5.53 (6.5, 1.40 1.73 1.46 1.92
2.23 1.81 2.25 5.53 (6.5, 1.40 1.74 1.44 1.91
0.95 s 0.94 s 2.21 m 0.88 d (6.5) 2.23 m 2.90 m
1.08 s 1.03 s 2.10 m 1.07 d (6.5) 2.33 m 2.82 m
1.13 s 0.89 s 1.60 m 0.97 d (6.5) 1.91 m
1.14 s 1.08 s 1.61 m 0.97 d (6.5) 1.91m
7.15 s
6.64 s
6.66 s
6.77 s 2.18 s
2.17 s
2.14 s
2.15 s
0.93 s 0.92 s 1.20 s
0.93 s 0.85 s 1.09 s
1.06 s 0.93 s 1.20 s
0.94 s 0.90 s 1.23 s
m m m m brs
1.60 1.83 2.36 2.58
8 m m m m
1.66 1.81 2.38 2.50
9 m m m m
1.91 m 1.35 m 1.62 m 1.50 m 2.07 m 1.35 m 1.73 m 1.82 m 1.96 m 1.50 m 0.73 s 1.11 s 1.73 m 1.02 d (6.4) 1.37 m 1.51 m 4.98 ddd (11.0,2.5,1.5) 6.99 d (1.5)
12
1.54 1.84 1.59 1.95 3.34
m m m m m dd 2.8) m m
0.87 s 1.00 s 2.42 m 0.80 d (6.5) 2.30 m 2.47 m
m m m m m m m dd 3.0)
m m m dd 3.0) m m m m
m m m dd 3.0) m m m m
2.02 m 7.30 s
6.61 m
1.91 s
2.00 s
2.48 s
1.23 s 1.08 s 0.73 s
1.08 s 1.08 s 1.02 s
0.83 s 0.73 s 1.70 s
2.57 m 2.82 m 2.83 m 1.19 d (7.1) 1.01 s 0.92 s 0.86 s
Please cite this article in press as: Wang, G.-W., et al. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.012
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G.-W. Wang et al. / Phytochemistry xxx (2015) xxx–xxx Table 2 C (125 MHz) NMR spectroscopic data for compounds 1–9 in CDCl3.
13
No.
1
2
3
4
5
6
7
8
9
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
36.0 t 34.6 t 217.8 s 47.4 s 51.2 d 19.3 t 26.2 t 135.1 s 133.2 s 36.8 s 21.0 t 30.7 t 44.6 s 49.9 s 30.9 t 28.0 t 50.8 d 15.9 q 18.6 q 33.7 d 18.6 q 40.6 t 78.9 d 149.7 d 129.4 s 174.4 s 10.6 q 26.1 q 21.2 q 24.2 q
36.7 t 34.8 t 217.1 s 47.6 s 53.4 d 27.6 t 22.4 t 41.7 d 147.1 s 39.0 s 116.0 d 37.1 t 44.4 s 47.0 s 33.7 t 27.9 t 51.3 d 14.5 q 22.0 q 33.3 d 18.3 q 40.6 t 78.9 d 149.6 d 129.4 s 174.4 s 10.6 q 25.5 q 21.7 q 18.3 q
34.2 t 34.0 t 219.1 s 46.9 s 52.4 d 22.9 t 121.8 d 148.1 s 45.2 d 35.8 s 20.5 t 33.0 t 44.2 s 52.0 s 33.9 t 28.6 t 52.7 d 22.5 q 23.1 q 35.1 d 20.6 q 120.1 d 147.1 s 134.0 d 130.0 s 171.1 s 10.7 q 28.0 q 21.1 q 27.1 q
35.5 t 28.1 t 77.5 d 38.8 s 48.5 d 23.1 t 121.6 d 148.8 s 48.4 d 35.8 s 22.8 t 33.4 t 43.5 s 52.7 s 34.7 t 28.5 t 53.3 s 24.7 q 17.1 q 35.9 d 18.4 q 35.0 t 25.5 t 142.4 d 127.8 s 169.3 s 12.6 q 29.5 q 23.7 q 30.8 q
34.8 t 27.2 t 79.5 d 38.6 s 43.3 d 23.1 t 120.5 d 136.4 s 52.7 d 34.9 s 33.4 t 25.3 t 51.5 s 152.5 s 115.2 d 44.9 t 50.4 s 28.3 q 16.4 q 34.0 d 16.8 q 46.3 t 208.9 s 46.6 t 34.5 d 180.8 s 15.7 q 22.3 q 19.2 q 16.9 q
29.2 t 25.1 t 75.8 d 36.5 s 37.9 d 22.9 t 122.3 d 146.0 s 51.2 d 34.5 s 27.7 t 118.6 d 155.9 s 49.7 s 36.5 t 37.7 t 46.1 s 25.2 q 15.0 q 38.7 d 21.4 q 48.0 t 204.3 s 150.4 s 126.6 d 175.0 s 14.7 q 24.0 q 22.2 q 27.5 q
34.0 t 33.4 t 219.8 s 46.8 s 53.7 d 22.6 t 121.3 d 146.5 s 43.9 d 36.2 s 18.1 t 25.6 t 52.0 s 50.4 s 39.6 t 120.6 d 156.0 s 26.7 q 23.0 q 28.6 d 20.6 q 51.5 t 201.1 s 134.2 d 139.2 s 171.9 s 13.9 q 25.1 q 21.3 q 28.3 q
36.6 t 29.2 t 211.2 s 46.6 s 38.1 d 23.1 t 122.7 d 145.7 s 50.5 d 34.7 s 25.2 t 118.5 d 156.1 s 50.0 s 36.6 t 38.3 t 36.9 s 25.2 q 14.0 q 37.8 d 17.8 q 40.1 t 107.3 s 147.7 d 131.5 s 172.4 s 10.3 q 28.1 q 22.0 q 22.9 q
36.7 t 29.2 t 76.7 d 46.6 s 38.1 d 23.1 t 122.8 d 145.7 s 50.5 d 34.7 s 25.2 t 118.6 d 156.2 s 50.1 s 36.7 t 38.3 t 36.9 s 25.2 q 17.8 q 37.8 d 22.0 q 40.2 t 107.2 s 147.6 d 131.5 s 172.3 s 10.3 q 28.1 q 22.0 q 22.9 q
determined by the PGME (phenylglycine methyl ester) method (Nagai and Kusumi, 1995; Handa et al., 2013). The (S)- and (R)PGME amides were obtained after 5 was treated with (R)- and (S)-PGME, respectively (Fig. 5). Thus, the absolute configuration of 25R for 5 could be elucidated according to the Dd values (Dd = dS dR) (Fig. 5). As such, compound 5 was defined as 3b-hydroxy-23-oxomariesia-7,14-dien-26-oic acid, and named abiesatrine L. Compound 6 was shown to have the molecular formula C30H44O4, as determined by the [M+H]+ ion peak at a m/z of 469.3237 in the positive HRESIMS, indicating a hydrogen deficiency index of nine. It had almost the same IR, 1H and 13C NMR spectroscopic data as those of the known compound 23-oxo-mariesiic acid B (Hasegawa et al., 1987b), except that CH3-27 was attached to C-24 due to the observation of the HMBC correlation of the methyl H3-27 [dH 2.18 (s)] with C-23 [dC 204.3 (s)], C-24 [dC 150.4 (s)] and C-25 [dC 126.6 (d)] (Fig. 6). In the NOESY spectrum, H-20 correlated with H3-30, indicating that C-17 in 6 had a S⁄ configuration. The a-orientation of the hydroxyl group at C-3 was established based on the NOESY correlation of H-3 with H3-19. Compound 6 was thus identified as 3a-hydroxy-23oxomariesia-7,12,24E-trien-26-oic acid and named abiesatrine M.
Compound 7 was assigned a molecular formula C30H42O4 from the [M H] ion peak at a m/z 465.3077 in negative HRESIMS, indicating a hydrogen deficiency index of ten. Compound 7 also showed similar 1H and 13C NMR spectra to those of the known compound 23-oxo-mariesiic acid B (Hasegawa et al., 1987b), except for the presence of a ketone moiety [dC 219.8 (s)] at the C-3 position of 7, instead of the 3-OH of 23-oxo-mariesiic acid B. This feature was verified by the HMBC correlations from H3-28 and H3-29 to the carbonyl carbon. The structure of compound 7 was then concluded to be 3,23-dioxomariesia-7,16,24E-trien26-oic acid, and given the name abiesatrine N. Neoabieslactone J (8) was deduced to have the molecular formula C30H42O4 from the [M+H]+ ion at a m/z of 467.3101 in positive HRESIMS. The 1H and 13C NMR spectra were quite similar to those of 23-hydroxy-3-oxo-9b-lanosta-7,24-dien-26,23-olide (Raldugin et al., 1989), except for the presence of an additional olefinic bond at C-12 and C-13 [dH 5.53 (dd); dC 118.5 (d), 156.1 (s)]. In the HMBC spectrum, H3-18 showed a long-range correlation with the olefinic carbon at dC 156.1 (C-13), and H-12 [dH 5.53 (dd)] was correlated with C-11 and C-13. These correlations suggested that the C-12 and C-13 positions of 8 accounted for a double bond. In the 1H and 13C NMR spectra of 8, typical resonance signals of the
Fig. 7. Key 1H–1H COSY, HMBC, and NOESY correlations for 9.
Please cite this article in press as: Wang, G.-W., et al. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.012
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Table 3 The inhibition of the isolated compounds from A. faxoniana on DNA topo II activity. Compounds
IC50 (lM)
Compounds
IC50 (lM)
Compounds
IC50 (lM)
1 2 3 4 5 6 7
>200 >200 53.5 >200 74.1 72.3 101.2
8 9 10 11 12 13 14
>200 >200 >200 >200 >200 >200 >200
15 16 17 18 19 Etoposide
>200 >200 >200 >200 >200 49.6
side-chain, including one hemiketal carbon at dC 107.3 (C-23), two olefinic carbon at dC 147.7 and 131.5 (C-24 and C-25), one ester carbonyl carbon at dC 172.4 (C-26), and one singlet methyl proton at dH 2.14 (s, H3-27), indicated that 8 has a c-lactone ring with a lactol as in neoabieslactone E (Li et al., 2009), 7,14,24-mariesatrien-26,23-olide-3a,23-diol (Gao et al., 2008), and 3a,23dihydroxylanosta-9(11),16,24-trien-26,23-olide (Handa et al., 2013). In the HMBC spectrum of 8, an olefinic proton at dH 6.64 (s, H-24) exhibited long-range correlations with C-23, C-26, and C-27. In the NOESY spectrum of 8, a methyl proton (CH3-27) was correlated with the olefinic proton (H-24). A methyl doublet (dH 0.97, 3H, J = 6.5 Hz, H-21) and its HMBC-correlated carbon signals [dC 36.9 (C-17), 37.8 (C-20), 40.1 (C-22)] were assigned to the side-chain moiety with a lactone ring. In the 1H and 13C NMR spectra of 8, some resonances showed broad weak peaks or minor peaks, pretty close to those of a type of previously reported lanostane triterpenoid featuring a c-lactone moiety with a lactol (Li et al., 2009; Handa et al., 2013). Gao et al. noted that these types of triterpenoids are usually tautomeric mixtures due to the C-23 epimerisation of the c-lactone moiety with a hemiketal (Gao et al., 2008). Therefore, the main resonance signals in the 1H and 13 C NMR spectra of 8 (Tables 1 and 2) were attributed to this phenomenon and were used to determine the structure of 8. On the basis of the above evidence, the structure of compound 8 was assigned as 23-hydroxy-3-oxo-9b-lanosta-7,12,24-trien-26, 23-olide. The molecular formula of C30H44O4 for compound 9 was determined from the [M H] ion at a m/z of 467.3236 in negative HRESIMS. The 1H and 13C NMR spectra of 9 showed similar broad weak peaks or minor peaks to those of 8, indicating that 9 possessed the same c-lactone moiety with a lactol as 8 and was also a tautomeric mixture due to C-23 epimerisation. Consequently, the structure of 9 was elucidated by the main resonance signals in the NMR spectrum. Analysis of the 1H and 13C NMR spectra exhibited that the NMR data of 9 was quite close to those of 8, except for the presence of a hydroxy group [dH 3.44 (t); dC 76.7 (d)] at C-3 in 9 instead of a ketone moiety as in 8. In the NOESY spectrum, H-3 was correlated with H-5 and H3-28, suggesting that the relative configuration of 3-OH was a b-configuration (Fig. 7). Consequently, the structure of compound 9 was elucidated to be 3b,23-dihydroxy-9b-lanosta-7,12,24-trien-26,23-olide, and it was named neoabieslactone K.
The isolated compounds were tested for their in vitro cytotoxic activities against the human tumour cell lines HepG2, Huh7, SMMC7721, HCT-116, MCF-7, and A549. Compound 3 demonstrated cytotoxicities against HCT-116, MCF-7, and A549 cells with IC50 values of 8.9, 7.6, and 4.2 lM, respectively. Meanwhile, compound 5 had IC50 values of 17.9, 20.1, and 25.3 lM for the human hepatoma cell lines HepG2, Huh7, and SMMC7721, respectively. Furthermore, the isolates were evaluated for their inhibitory effects on human DNA topos I and II in vitro. The conversion of supercoiled pHOT-1 DNA to relaxed DNA by topos I and II was examined in the presence of these compounds. None of these compounds inhibited DNA relaxation by topo I, even when the concentrations were increased up to 200 lM. However, compounds 3, 5, 6, and 7 showed complete inhibition of topo II relaxation activity at 200 lM. Compounds 8 and 9 showed 18% and 9% inhibition, respectively, against topo II activity at 200 lM. Compounds 3, 5, and 6 had inhibitory effects against topo II with IC50 values of 53.5, 74.1, and 72.3 lM, respectively, which were comparable to that of the topo II inhibitor etoposide (IC50 = 49.6 lM) (Table 3). 3. Conclusions From the branches and leaves of A. faxoniana, 9 new (1–9) and 10 known triterpenoids were isolated. Compounds 1–3 have a a,b-unsaturated-c-lactone ring in the C-17 side-chain. Compounds 8 and 9 were obtained as epimeric mixtures potentially arising from tautomerism at C-23 of the c-lactone structure. A plausible biogenetic pathway of the lactone structure in compounds 1–3, and 8–9 is shown in Fig. 8. These compounds might originate from the corresponding 23-keto type lanostanes. Their biosynthesis might provide some insight into the structural transformation of lanostane-type triterpenoids. Since triterpenoids from some Abies plants displayed cytotoxic and antitumor effects, the isolated compounds were tested for their cytotoxic activities and DNA topoisomerase inhibitory effects. Compound 3 showed cytotoxic activities against HCT-116, MCF-7, and A549 cell lines. It also had inhibitory effect against topo II with IC50 value of 53.5 lM. Further evaluation of the inhibition mechanism by compound 3 is now in progress. 4. Experimental 4.1. General experimental procedures 1D and 2D NMR spectrum were determined with a Bruker Avance-500 spectrometer. ESIMS were acquired on an Agilent LC/MSD Trap XCT micromass spectrometer, whereas HRESIMS were measured using an Agilent 6520 Accurate-Mass Q-TOF LC/MS. Optical rotations were obtained with a JASCO P-2000 polarimeter. UV spectrum were obtained on a Shimadzu UV2550 spectrometer. IR spectrum were recorded on a Bruker FTIR Vector 22 spectrometer using KBr pellets. Column chromatography
Fig. 8. Postulated biosynthetic pathway of the lactone structure in the side-chain.
Please cite this article in press as: Wang, G.-W., et al. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.012
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(CC) was performed on silica gel (100–200, 200–300 mesh, Shandong, China), Sephadex LH-20 (GE Healthcare, Sweden) and YMC 50 lm ODS-A (Milford, MA, USA), respectively. Preparative TLC (0.4–0.5 mm, 20 20 cm) was conducted with glass plates precoated with silica gel GF254 (Yantai, Shandong, China). A semi-preparative column (Agilent ZORBAX SB-C18, 5 lm, 9.4 250 mm) and analytical column (Agilent ZORBAX ExtendC18, 5 lm, 4.6 250 mm) were used for HPLC (Shimadzu LC-2010A HT). 4.2. Plant material The branches and leaves of A. faxoniana were collected from Li county, Sichuan province, in August 2009 and authenticated by Prof. Han-Ming Zhang in the Department of Pharmacognosy, Second Military Medical University. A voucher specimen (20090813001) was deposited at the Herbarium of the School of Pharmacy, Second Military Medical University, Shanghai, People’s Republic of China. 4.3. Extraction and isolation Air-dried branches and leaves of A. faxoniana (5.3 kg) were powdered and extracted with EtOH:H2O (80:20, v/v) three times under conditions of reflux. The solvent was removed in vacuo to afford a crude EtOH extract, which was suspended in H2O and then successively partitioned with petroleum ether (PE), CH2Cl2, EtOAc, and nBuOH. The CH2Cl2 extract (100.0 g) was subjected to silica gel (/ 8 100 cm; 100–200 mesh, 1000 g) CC and eluted with a PE/EtOAc gradient (50:1–1:1) to give 10 fractions (Fr.1–Fr.10) based on TLC analysis. Fr.5 [PE/EtOAc (10:1), 30.0 g] underwent CC (/ 4.5 60 cm) on silica gel (200–300 mesh, 700 g) and was eluted with a gradient of PE/EtOAc (20:1–1:1) to afford 8 subfractions (Fr.5-1–Fr.5-8). Subfraction Fr.5-3 [PE/EtOAc (10:1), 4.0 g] was subjected to semi-preparative HPLC (MeOH/H2O, 50:50) to give 7 (40.0 mg), 18 (20.0 mg), and 19 (30.0 mg). Fr.6 [PE/EtOAc (8:1), 28.0 g] was applied to a silica gel column (/ 4.5 60 cm; 200–300 mesh, 600 g) and was eluted with a PE/EtOAc gradient (10:1–1:1) to give 8 subfractions (Fr.6-1–Fr.6-8). From subfraction Fr.6-5 [PE/EtOAc (5:1), 3.0 g], compounds 1 (50.0 mg), 9 (25.0 mg), 13 (50.0 mg), and 14 (28.0 mg) were isolated after CC over Sephadex LH-20 (/ 4.0 150 cm; MeOH) followed by semipreparative HPLC (MeOH/H2O, 50:50). Fr.6–7 [PE/EtOAc (3:1), 3.6 g] was subjected to CC on silica gel (/ 2.5 30 cm; 50 g, 200–300 mesh) and was eluted with a PE/EtOAc gradient (10:1– 1:1) followed by semi-preparative HPLC (MeOH/H2O, 50:50) to give 2 (12.0 mg), 5 (24.6 mg), and 6 (15.4 mg). Compound 10 (40.0 mg) was crystallised from Fr.6–6 [PE/EtOAc (4:1), 1.6 g]. Compounds 11 (30.0 mg) and 12 (25.0 mg) were crystallised from Fr.6-8 [PE/EtOAc (1:1), 2.0 g]. Fr.6-4 [PE/EtOAc (6:1), 2.2 g] was subjected to CC on silica gel (/ 2.5 30 cm; 50 g, 200–300 mesh) and was eluted with a CH2Cl2/MeOH gradient (10:1–1:1) to give 5 subfractions (Fr.6-4a–Fr.6-4e). Fr.6–4e [CH2Cl2/MeOH (1:1), 0.6 g] was subjected to semi-preparative HPLC (MeOH/H2O, 50:50) to give 3 (10.0 mg), 15 (16.0 mg), and 16 (15.0 mg) and was then further eluted with (MeOH/H2O, 60:40) to yield 4 (50.0 mg), 8 (18.0 mg), and 17 (20.0 mg). 4.3.1. Neoabieslactone G (1) Amorphous powder; [a]20 D +51 (c 0.1, CHCl3); UV (MeOH) kmax (log e) 206.9 (3.10) nm; IR (KBr) mmax 3070, 3022, 2930, 2852, 1741, 1698, 1646, 1460, 1410, 1378, 1360, 1197, 1095, 1040, 933, 878, 818 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (positive) m/z 453.3302 [M+H]+ (calcd for C30H45O3, 453.3290).
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4.3.2. Neoabieslactone H (2) Amorphous powder; [a]20 D +29 (c 0.1, CHCl3); UV (MeOH) kmax (log e) 208.0 (3.90) nm; IR (KBr) mmax 3069, 3020, 2928, 2852, 1740, 1698, 1648, 1460, 1410, 1378, 1360, 1197, 1095, 1040, 933, 878, 818 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (positive) m/z 453.3296 [M+H]+ (calcd for C30H45O3, 453.3290). 4.3.3. Neoabieslactone I (3) Amorphous powder; [a]20 34 (c 0.1, CHCl3); UV (MeOH) kmax D (log e) 278.0 (3.60) nm; IR (KBr) mmax 3069, 3020, 2928, 2852, 1745, 1696, 1648, 1646, 1460, 1410, 1378, 1360, 1197, 1095, 1040, 933, 878, 818 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (positive) m/z 451.3127 [M+H]+ (calcd for C30H43O3, 451.3134). 4.3.4. Abiesatrine K (4) Amorphous powder; [a]20 D +53 (c 0.1, CHCl3); UV (MeOH) kmax (log e) 205.0 (2.80) nm; IR (KBr) mmax 3445, 2925, 1772, 1698, 1646, 1465, 1378, 1045, 818 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (negative) m/z 455.3610 [M H] (calcd for C30H47O3, 455.3603). 4.3.5. Abiesatrine L (5) Amorphous powder; [a]20 D +54 (c 0.1, CHCl3); UV (MeOH) kmax (log e) 230.0 (3.80) nm; IR (KBr) mmax 3345, 2928, 1769, 1698, 1646, 1460, 1410, 1378, 1360, 1040, 933, 878 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (positive) m/z 471.3383 [M+H]+ (calcd for C30H47O4, 471.3396). 4.3.6. Abiesatrine M (6) Amorphous powder; [a]20 100 (c 0.1, CHCl3); UV (MeOH) kmax D (log e) 296.0 (4.12) nm; IR (KBr) mmax 3345, 2928, 1769, 1698, 1648, 1646, 1460, 1410, 1378, 1360, 1040, 878 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (positive) m/z 469.3237 [M+H]+ (calcd for C30H45O4, 469.3240). 4.3.7. Abiesatrine N (7) Amorphous powder; [a]20 D +113 (c 0.1, CHCl3); UV (MeOH) kmax (log e) 236.1 (4.01) nm; IR (KBr) mmax 2928, 1769, 1698, 1648, 1646, 1460, 1410, 1378, 1360, 1040, 878 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (negative) m/z 465.3077 [M H] (calcd for C30H41O4, 465.3083). 4.3.8. Neoabieslactone J (8) Amorphous powder; [a]20 D +16 (c 0.1, CHCl3); UV (MeOH) kmax (log e) 210.0 (4.71) nm; IR (KBr) mmax 3419, 2917, 1754, 1700, 1437, 1207, 954, 705 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (positive) m/z 467.3101 [M+H]+ (calcd for C30H43O4, 467.3083). 4.3.9. Neoabieslactone K (9) Amorphous powder; [a]20 12 (c 0.1, CHCl3); UV (MeOH) kmax D (log e) 212.1 (4.65) nm; IR (KBr) mmax 3453, 2995, 2914, 1755, 1463, 1311, 1054, 954, 700 cm 1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS (negative) m/z 467.3236 [M H] (calcd for C30H43O4, 467.3240). 4.3.10. (S)- and (R)-PGME amides of 5 To 5 (2.0 mg) in N,N-dimethylformamide (DMF, 0.5 mL) was added (S)-PGME (5.0 mg). Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop, 10.0 mg), 1-hydroxybenzotriazole (HOBT, 4.0 mg), and N-methylmorpholine (30 lL) were then successively added. The reaction mixture was moved to a refrigerator at 4 °C overnight. The mixture was then stirred for 2 h at room temperature and subjected to HPLC
Please cite this article in press as: Wang, G.-W., et al. Lanostane-type triterpenoids from Abies faxoniana and their DNA topoisomerase inhibitory activities. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.04.012
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(Agilent ZORBAX SB-C18, 5 lm, 9.4 250 mm; solvents CH3CN– 0.2%CF3CO2H, 90:10; detector, UV 210 nm), yielding the (S)-PGME amide of 5 (0.8 mg). The same procedure was used to prepare the (R)-PGME amide of 5 (0.8 mg). 4.3.11. (S)-PGME amide of 5 Amorphous powder; ESI-MS m/z 618 [M+H]+, 1H NMR (500 MHz, CDCl3) dH 5.53 (1H, dd, J = 6.5, 3.0 Hz, H-7), 5.14 (1H, dd, J = 1.2, 2.8 Hz, H-15), 0.80 (3H, s, H-18), 1.00 (3H, s, H-19), 2.35 (1H, m, H-20), 0.73 (3H, d, J = 6.5 Hz, H-21), 2.22 (1H, m, H22), 2.37 (1H, dd, J = 14.1, 1.8 Hz, H-22), 2.53 (1H, m, H-24), 2.83 (1H, m, H-24), 2.83 (1H, m, H-25), 1.29 (3H, d, J = 7.1 Hz, H-27), 1.01 (3H, s, H-28), 0.92 (3H, s, H-29), 0.78 (3H, s, H-30). 4.3.12. (R)-PGME amide of 5 Amorphous powder; ESI-MS m/z 618 [M+H]+, 1H NMR (500 MHz, CDCl3) dH 5.56 (1H, dd, J = 6.5, 3.0 Hz, H-7), 5.18 (1H, dd, J = 1.2, 2.8 Hz, H-15), 0.87 (3H, s, H-18), 1.00 (3H, s, H-19), 2.42 (1H, m, H-20), 0.80 (3H, d, J = 6.5 Hz, H-21), 2.30 (1H, m, H22), 2.47 (1H, dd, J = 14.1, 1.8 Hz, H-22), 2.57 (1H, m, H-24), 2.82 (1H, m, H-24), 2.83 (1H, m, H-25), 1.19 (3H, d, J = 7.1 Hz, H-27), 1.01 (3H, s, H-28), 0.92 (3H, s, H-29), 0.86 (3H, s, H-30).
4.6. DNA topoisomerase I inhibitory activity assay Reactions were carried out in the same manner as described for the topo II relaxation assays, except that the reaction mixtures contained 10 mM Tris–HCl (pH = 7.9), 1 mM EDTA, 150 mM NaCl, 0.1% BSA, 0.1 mM spermidine, 5% glycerol, and supercoiled pHOT-1 DNA (0.25 lg). Acknowledgments We thank Ms. Yangyun Zhou of Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, PR China, for assisting with DNA topoisomerase inhibitory activities. The work was supported by program NCET Foundation, National Natural Science Foundation of China (NSFC, 81230090), Shanghai Leading Academic Discipline Project (B906), Key laboratory of drug research for special environments, PLA, Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300), the Scientific Foundation of Shanghai China (12401900801, 13401900101), National Major Project of China (2011ZX09307-002-03) and the National Key Technology R&D Program of China (2012BAI29B06). Appendix A. Supplementary data
4.4. Cytotoxic assay Cell viability was determined by a MTT assay (Yang et al., 2008b). Briefly, cells were seeded in 96-well plates at 6 103 cells/well and exposed to the test compounds (0, 1, 5, 10, 25, and 50 lM) for 24 h. The cultures were treated with DMSO as the vehicle control. After 24 h of treatment, MTT solution 10 lL (5 mg/mL; Sigma; St. Louis, MO) was added to each well, and the plates were incubated for 2–4 h at 37 °C. The supernatant was then removed from the formazan crystals, and DMSO 100 lL was added to each well. The absorbance at 570 nm was measured using an OPTImax microplate reader. The cell viability was calculated by dividing the mean optical density (OD) of compound-containing wells by that of the DMSO-control wells. 4.5. DNA topoisomerase II inhibitory activity assay Supercoiled pHOT-1 DNA, 5X universal stop buffer, human DNA topos I (recombinant) and IIa (p170 form) were purchased from TopoGen, Inc. (Columbus, OH). The DNA topo inhibitors etoposide and camptothecin were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). The topo II activity was measured by assessing the relaxation of supercoiled pHOT-1 DNA (Muller et al., 1988; Wada et al., 2001). The reaction mixture contained 50 mM Tris–HCl (pH = 8.0), 150 mM NaCl, 10 mM MgCl2, 5 mM ATP, 0.5 mM dithiothreitol, 30 lg bovine serum albumin (BSA)/mL, supercoiled pHOT-1 DNA (0.2 lg), the indicated drug concentrations (1% DMSO), and 1 U of topo IIa in a final volume of 20 lL. The reaction mixtures were incubated for 30 min at 37 °C and were then terminated with 5 stop buffer (5 lL per 20 lL reaction volume). The stop buffer contained 5% sarkosyl, 0.0025% bromophenol blue, and 25% glycerol. The reaction products were electrophoresed on a 1% agarose gel in TAE (tris–acetate–EDTA) running buffer at 8 V/cm for 1 h. The gels were stained with ethidium bromide (0.5 lg/mL) for 1 h and destained in water for 30 min. For the quantitative analysis of the topo II activity, the gels were directly scanned with a FLA2000 fluoroimage analyser, and the area representing supercoiled DNA was calculated. The IC50 values were determined by interpolation from plots of the topo II activity versus the inhibitor concentration.
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