Note pubs.acs.org/jnp
Cite This: J. Nat. Prod. 2018, 81, 418−422
Modified Abietane Diterpenoids from Whole Plants of Selaginella moellendorffii Lei-Yu Ke,†,‡,§,# Yu Zhang,†,# Meng-Yuan Xia,†,‡ Jing-Xian Zhuo,† Yue-Hu Wang,*,†,‡ and Chun-Lin Long*,⊥ †
Key Laboratory of Economic Plants and Biotechnology and Yunnan Key Laboratory for Wild Plant Resources, State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China ‡ Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences, Yezin, Nay Pyi Taw 05282, Myanmar § University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China ⊥ College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, People’s Republic of China S Supporting Information *
ABSTRACT: A new modified abietane diterpenoid, (3S,4S,5R,10S)-18(4→ 3)-abeo-3,4,12,18-tetrahydroxy-8,11,13-abietatrien-7-one (1), and two novel dimers, selaginedorffones A (2) and B (3), featuring a new cyclohexene moiety that was biogenetically constructed from two modified abietane diterpenoids through a Diels−Alder reaction were obtained from a methanolic extract of Selaginella moellendorffii, a traditional Chinese herb. The structures of 1−3 were identified by a combination of NMR spectroscopic analysis and ECD calculations. In the present study, diterpenoids were identified from S. moellendorffii for the first time, which supports the presence of diterpene synthases in this plant. These three diterpenoids (1−3) were evaluated for their growth-inhibitory activities against several human cancer cell lines. Of these substances, selaginedorffone B (3) showed cytotoxicity against the MCF7 human-breast-cancer-cell line (IC50 9.0 μM). hydroxy-group (3429 cm−1), conjugated-carbonyl-group (1635 cm−1), and phenyl-ring (1602, 1500, and 1454 cm−1) absorption peaks. The 1H NMR data (Table 1) indicated a tetrasubstituted phenyl ring [δH 7.76 (1H, s) and 6.72 (1H, s)] and four methyl groups [δH1.35 (3H, s), 1.30 (3H, s), 1.20 (3H, d, J = 6.8 Hz), and 1.18 (3H, d, J = 6.9 Hz)]. As shown in Table 1, the 13C NMR data exhibited 20 carbon signals, consisting of four methyls, four methylenes, four methines, six quaternary carbons, an oxygenated sp2 tertiary carbon (δC 162.7), and a ketocarbonyl carbon (δC 201.1). The characteristic NMR signals described above suggest that 1 is an abietane diterpenoid with aromatization of ring C.24 By analysis of the 1H−1H COSY correlations of compound 1 (Figure 1), the presence of three spin systems, C-1−C-2, C-5− C-6, and C-16−C-15−C-17 was revealed. According to the HMBC correlations from H3-18 to C-2, C-3, and C-4; H2-19 to C-3 and C-5; and H3-20 to C-1, C-5, C-9, and C-10, ring A could be constructed, and an 18(4→3)-abeo-abietane skeleton for 1 was proposed. The HMBC spectrum of 1 displayed correlations from H2-6 to C-7; H-11 to C-8 and C-13; H-14 to C-7, C-9, C-12, and C-15; and H3-16 and H3-17 to C-13, revealing the presence of rings B and C in 1; the planar
Selaginella moellendorffii Hieron. (Selaginellaceae) is a perennial herb distributed in mainland China, Japan, the Philippines, and Vietnam.1 The whole plant of S. moellendorffii is used to treat several conditions in traditional Chinese folk medicine.2,3 Previous investigations on this plant have led to reports on alkaloids,3,4 lignans and neolignans,5−9 phenylpropanoids,7,8 flavonoids and biflavonoids,7,10−20 anthraquinones,16 a selaginellin,21 a monoterpene glucoside,13 a coumarin,22 and an isochroman-1-one.18 Among these compounds, ginkgetin was reported to possess cytotoxicity against the OVCAR-3 humanovarian-adenocarcinoma-cell line,10 selaginellic acid showed antiplatelet-aggregation activity,23 and selaginellin S exhibited inhibitory activity against hepatitis B virus gene expression and replication.21 In the present study, further work was conducted to obtain structurally novel constituents of S. moellendorffii.3,8,9,13 As a result, a new modified diterpenoid (1) and two novel dimers (2 and 3) were isolated and identified. The cytotoxic activities of these three modified abietane diterpenoids were evaluated against a small panel of human-cancer cell lines. Compound 1, a white powder with seven degrees of unsaturation, gave a molecular formula of C 20 H 28 O 5 , determined on the basis of 13C NMR data (Table 1) and its HRESIMS peak at m/z 371.1853 [M + Na]+ (calcd for C20H28NaO5, 371.1834). The IR spectrum of 1 showed © 2018 American Chemical Society and American Society of Pharmacognosy
Received: October 30, 2017 Published: February 7, 2018 418
DOI: 10.1021/acs.jnatprod.7b00909 J. Nat. Prod. 2018, 81, 418−422
Journal of Natural Products
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Table 1. 1H and 13C NMR Data of 1 in Methanol-d4 (δ in ppm, J in Hz) no.
δH (800 MHz)
δC (201 MHz)
1α 1β 2α 2β 3 4 5 6α 6β 7 8 9 10 11 12 13 14 15 16 17 18 19
1.75 ddd (13.9, 13.9, 3.2) 2.16 ddd (13.9, 3.2, 3.2) 1.52 ddd (13.9, 3.2, 3.2) 2.26 ddd (13.9, 13.9, 3.2)
35.2
20
2.23 dd (13.5, 3.8) 2.62 dd (18.6, 3.8) 2.86 dd (18.6, 13.5)
6.72 s
7.76 s 3.21 m 1.18 d (6.9) 1.20 d (6.8) 1.30 s 3.73 d (11.0) 3.61 d (11.0) 1.35 s
34.4 75.2 77.0 43.8 36.6 201.1 123.9 157.5 38.3 110.4 162.7 134.8 127.2 27.7 22.8 22.7 23.2 65.6
Figure 2. Comparison of the experimental and calculated ECD spectra of 1.
HRESIMS peak, which occurred at m/z 643.3031 [M + Na]+ (calcd for C40H44NaO6, 643.3036), requiring 19 indices of hydrogen deficiency. Its 1H NMR data (Table 2) indicated the presence of two tetrasubstituted phenyl rings [δH 7.93 (1H, s), 7.88 (1H, s), 6.95 (1H, s), and 6.68 (1H, s)], an exocyclic double bond [δH 5.27 (1H, s) and 5.06 (1H, s)], and six methyl groups [δH 1.43 (3H, s), 1.23 (3H, d, J = 7.2 Hz), 1.23 (3H, d, J = 6.9 Hz), 1.22 (3H, d, J = 7.2 Hz), 1.21 (3H, d, J = 6.9 Hz), and 1.01 (3H, s)]. As shown in Table 2, the 13C NMR data of 2 showed 40 signals for 6 methyls, 7 methylenes, 9 methines, 13 quaternary carbons, 2 oxygenated sp2 tertiary carbons (δC 162.7 and 161.5), and 3 ketocarbonyl groups (δC 211.4, 198.3, and 187.0). Comparison of its 1H and 13C NMR spectra with those of 1 revealed 2 to be a diterpenoid dimer. On the basis of the 2D NMR correlations (Figure 1) in the 1H−1H COSY and HMBC spectra (Supporting Information) of 2, rings A−C and A′−C′ were readily constructed. The HMBC correlations observed from H-18β to C-4, H-18α to C-3′, H-18′β to C-4, H18′α to C-2′, H-19α to C-2′, and H-19β to C-3 indicated the presence of a new cyclohexene moiety (ring D) in 2, consisting of C-3−C-4−C-19−C-3′−C-18′−C-18. Thus, the planar structure of 2 (Figure 1) was determined to be a modified abietane diterpenoid dimer. The ROESY correlations (Figure 1) between H-1β (δH 2.49) and H3-20 indicated that these protons were cofacial, and they
24.8
structure of compound 1 was then elucidated as 18(4→3)-abeo3,4,12,18-tetrahydroxy-8,11,13-abietatrien-7-one. The ROESY correlations (Figure 1) between H-1β (δH 2.16) and H3-20 indicated that these protons were cofacial, and these were assigned arbitrarily as β-oriented. In turn, the ROESY correlations of H3-18/H-1α, H3-18/H-5, and H2-19/H-5 indicated that these protons were α-oriented. The absolute configuration of 1 (Figure 2) was established as 3S,4S,5R,10S by comparisons of the experimental and calculated ECD spectra of 1. Accordingly, the structure of 1 was determined to be (3S,4S,5R,10S)-18(4→3)-abeo-3,4,12,18-tetrahydroxy8,11,13-abietatrien-7-one. Selaginedorffone A (2) gave the molecular formula C40H44O6, on the basis of its 13C NMR data (Table 2) and
Figure 1. Key 2D NMR correlations of compounds 1 and 2. 419
DOI: 10.1021/acs.jnatprod.7b00909 J. Nat. Prod. 2018, 81, 418−422
Journal of Natural Products
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Table 2. 1H and 13C NMR Data of 2 and 3 in Methanol-d4 (δ in ppm, J in Hz) 2 no.
δH (800 MHz)
1α
1.90 dd (14.6, 6.0)
42.5
1β 2 3 4 5 6α 6β
2.49 d (14.6) 4.25 br d (6.0)
7 8 9 10 11 12 13 14 15 16 17 18α 18β 19α 19β 20 1′α 1′β 2′ 3′ 4′ 5′ 6′α 6′β 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 17′ 18′α 18′β 19′a 19′b 20′ a
3 δC (201 MHz)
6.23 s
6.95 s
7.88 s 3.26 m 1.23 d (6.9)a 1.23 d (7.2)a 2.29 m 3.28 m 2.62 br d (16.2) 3.11 br d (16.2) 1.43 s 3.06 d (12.7) 2.77 d (12.7)
3.73 br d (13.5) 2.70 dd (17.3, 3.5) 2.84 dd (17.3, 13.5)
6.68 s
7.93 s 3.26 m 1.22 d (7.2)a 1.21 d (6.9)a 1.89 m 2.31 m 5.27 s 5.06 s 1.01 s
19/H-5′ were observed, implying that the C-3′−C-2′ bond was β-oriented and the C-3′−C-4′ bond was α-oriented. Furthermore, the ROESY correlations of H-2/H-18α, H18′α/H-19α, H-5′/H2−19, and H-5′/H-1′α, along with H18β/H-18′β and H-1′β/H3−20′, were used to establish the configurations of these protons. Therefore, the relative configuration of 2 was elucidated as shown. The molecular formula of selaginedorffone B (3), C40H46O6, was determined on the basis of its 13C NMR data (Table 2) and pseudomolecular ion peak at m/z 645.3193 [M + Na]+ (calcd for C40H46NaO6, 645.3192) in the HRESIMS, which required 18 indices of hydrogen deficiency. A detailed comparison of its 1 H and 13C NMR data (Table 2) with those of 2 revealed that these two compounds were close analogues, but compound 3 was found to have one less double bond than 2. According the 1 H−1H COSY and HMBC correlations of 3 (Figure S1, Supporting Information), compound 3 was deduced as being the 5,6-dihydro analogue of 2. The relative configuration of 3 was shown to be similar to that of 2 from the observed ROESY correlations (Figure S1, Supporting Information) and from the coupling constants of H-1α and H-2 (J1α,2 = 5.7 Hz). Moreover, H-5 could be assigned as α-oriented according to the correlations of H-5/H-1α and H3-20/H-1β observed in the ROESY spectrum (Supporting Information). The absolute configurations 2S,10R,3′S,5′S,10′S and 2S,5S,10S,3′S,5′S,10′S were determined for 2 (selaginedorffone A) and 3 (selaginedorffone B), respectively, by comparisons of the experimental and calculated ECD spectra (Figure S2, Supporting Information). As shown in Scheme 1, a plausible biogenetic pathway of the modified abietane diterpenoids, 1−3, is suggested. Miltiradiene, a product synthesized in vitro by miltiradiene synthase (SmMDS) cloned from S. moellendorffii,27 may be transformed by several steps of oxidation into margocilin, a diterpenoid ketone with an aromatization of ring C. Margocilin may then be transformed into iii with an 18(4→3)-abeo-modified abietane diterpenoid skeleton as a result of Wagner−Meerwein rearrangement procedures.28 The common intermediate, iv, could be generated after the oxidation of iii. Oxidation of the double bond at C-3 of iv would yield compound 1, whereas oxidation of the C-2 after a double-bond migration from C-3− C-4 to C-3−C-18 and dehydration of iv would yield two key intermediates, vii and viii. Through a Diels−Alder reaction, the diene vii and the dienophile viii would yield compound 3, and its dehydrogenation would yield compound 2. The genome of S. moellendorffii has been sequenced, and it expresses many putative diterpene synthases.29 Two bifunctional diterpene synthases from S. moellendorffii, miltiradiene synthase (SmMDS) and SmCPSKSL1, were used to generate in vitro miltiradiene and λ-7,13E-dien-15-ol, respectively, from geranylgeranyl diphosphate (GGDP).27,30 However, diterpenoids have not been reported in S. moellendorffii thus far in the literature. Compounds 1−3 were tested for the growth-inhibitory activities against SW480 human rectal-cancer, HL-60 human leukemia, SMMC-7721 human liver-cancer, MCF-7 human breast-cancer, and A-549 human lung-cancer cell lines. Selaginedorffone B (3) was the only cytotoxic compound found (MCF-7 cells, IC50 value of 9.0 μM). Compound 3 was inactive against the SMMC-7721, SW480, HL-60, and A-549 cell lines, whereas compounds 1 and 2 were inactive against all five cancer cell lines used.
68.2 143.9 125.6 164.9 120.4
187.0 123.2 152.8 39.2 112.2 161.5 135.9 125.7 27.9 22.8b 22.8b 28.3 37.8 32.4 50.5 211.4 56.5 153.7 43.6 39.0
198.3 123.9 152.9 42.1 111.2 162.7 136.1 127.6 28.0 22.8b 22.7b 27.4 111.4 22.2
δH (800 MHz) 1.90 dd (14.0, 5.7) 2.50 d (14.0) 4.13 br d (5.7)
2.64 br d (15.0) 2.83 d (17.4) 2.39 dd (17.4, 15.0)
6.81 s
7.77 s 3.21 m 1.17 d (6.8) 1.19 d (6.8) 2.13 m 2.94 m 2.41 br d (17.0) 2.57 br d (17.0) 1.16 s 3.15 d (13.1) 2.84 d (13.1)
3.61 br d (13.4) 2.59 br d (17.2) 2.80 dd (17.2, 13.4)
6.74 s
7.91 s 3.26 m 1.24 d (6.8) 1.22 d (6.8) 1.87 m 2.21 m 5.29 s 4.99 s 1.01 s
δC (201 MHz) 43.1
68.5 132.2 127.5 45.1 38.3
199.6 123.9 155.8 36.5 110.7 162.5 134.9 127.7 27.8 22.8 22.8 26.7 36.7 23.4 50.8 212.0 56.3 151.7 43.7
198.5 123.8 153.2 41.7 111.4 162.7 135.9 127.5 27.9 22.7 22.7 26.7 111.5 22.2
Interchangeable. bInterchangeable.
were arbitrarily assigned as β-oriented. By comparison of the J1,2 value of compound 2 (J1,2 = 6.0 Hz) with those of the 2βhydroxy (J1,2 = 5.9 Hz) and 2α-hydroxy (J = 13.6 Hz) analogues,25,26 the H-2 proton in 2 was determined to be αoriented. Crucial ROESY correlations of H2-18/H-19′a and H2420
DOI: 10.1021/acs.jnatprod.7b00909 J. Nat. Prod. 2018, 81, 418−422
Journal of Natural Products
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Scheme 1. Plausible Biogenetic Route of 1−3
■
UV (MeOH) λmax (log ε) 397 (1.78), 284 (3.61), 231 (3.75), 206 (3.86) nm; ECD (c 0.042, MeOH) λmax (Δε) 323 (+3.91), 294 (−2.63), 215 (+0.78), 218 (+0.70), 209 (−0.95) nm; IR (KBr) νmax 3429, 1635, 1602, 1500, 1454, 1384, 1304, 1178, 1107, 1072 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 371 [M + Na]+, HRESIMS m/z 371.1853 [M + Na]+ (calcd for C20H28NaO5, 371.1834). Selaginedorffone A (2). White powder; [α]22.1D + 9.5 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 291 (3.99), 231 (4.09), 206 (4.37) nm; ECD (c 0.027, MeOH) λmax (Δε) 353 (−0.68), 318 (+0.92), 279 (−0.72), 214 (+3.13) nm; 1H and 13C NMR data, see Table 2; ESIMS m/z 643 [M + Na]+, HRESIMS m/z 643.3031 [M + Na]+ (calcd for C40H44NaO6, 643.3036). Selaginedorffone B (3). White powder; [α]22.4D + 73.3 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 286 (4.12), 232 (4.24), 206 (4.38) nm; ECD (c 0.021, MeOH,) λmax (Δε) 322 (+2.80), 241 (−1.86), 204 (+5.61) nm; 1H and 13C NMR data, see Table 2; ESIMS m/z 621 [M − H]−, HRESIMS m/z 645.3193 [M + Na]+ (calcd for C40H46NaO6, 645.3192). Cytotoxicity Assay. Compounds 1−3 were tested in vitro for their cytotoxicities using the SW480 human rectal-cancer, A-549 human lung-cancer, SMMC-7721 human liver-cancer, MCF-7 human breast-cancer, and HL-60 human leukemia cell lines, in a 3-(4,5dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)2H-tetra-olium (MTS; Promega, Beijing, People’s Republic of China) assay.31 The details are presented in the Supporting Information. Paclitaxel (Sigma, St. Louis, MO) was used as a positive control. The cytotoxicity for each compound was expressed as an IC50 value by the Reed and Muench method.32 The IC50 value of paclitaxel was
