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Bioactive Enmein-Type ent-Kaurane Diterpenoids from Isodon phyllostachys Jin Yang,†,‡,⊥ Wei-Guang Wang,†,⊥ Hai-Yan Wu,†,‡ Xue Du,† Xiao-Nian Li,† Yan Li,† Jian-Xin Pu,*,† and Han-Dong Sun*,† †

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 ‡ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Thirty-two enmein-type ent-kaurane diterpenoids, including 13 new compounds, were isolated from the aerial parts of Isodon phyllostachys. Compounds 1 and 2 are the first examples of 3,20:6,20-diepoxyenmein-type ent-kauranoids, and the structures of these new compounds were established mainly by analyzing NMR and HREIMS data. The absolute configurations of 1 and 8 and the relative configuration of 9 were determined using single-crystal X-ray diffraction. Compounds 11, 15, 20, and 21 were active against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480), with IC50 values ranging from 1.2 to 5.0 μM. Compounds 3, 11, 15, 17, 20, 21, 25, and 29 strongly inhibited NO production in LPS-stimulated RAW264.7 cells, with IC50 values ranging from 0.74 to 4.93 μM. investigated. Consequently, 13 new enmein-type ent-kauranoids, phyllostacins J−M (1−4), 20-epiphyllostacin M (5), 11epitaibaijaponicain A (6), 11,17-diepitaibaijaponicain A (7), phyllostacin N (8), 20-epiphyllostacin N (9), 17-epidihydroisodocarpin (10), 20-episerrin C (11), phyllostacin O (12), and 20-epiphyllostacin O (13), together with 19 known analogues, 15α,20β-dihydroxy-6β-methoxy-6,20-epoxy-6,7-seco-ent-kaur16-en-1,7-olide (14),16 serrin C (15),17 dihydroisodocarpin (16),18 nodosin (17),19 rabdosichuanin C (18),20 nervosin (19),21 isodocarpin (20),18 serrin B (21),17 irroratin A (22),22 phyllostacin B (23),9 phyllostacin A (24),9 enmein (25),23 dihydroenmein (26),24 longirabdolide C (27),25 sculponeatin B (28),26 sculponeatin A (29),26 ludongnin E (30),27 sculponin G (31),28 and sculponin D (32),28 were obtained. This is the first time that such a large number of enmein-type diterpenoids have been obtained from a single plant. Compounds 1 and 2 are the first examples of 3,20:6,20-diepoxy enmein-type entkauranoids, and compound 3 contains a rare 6,20:11,20diepoxy group. Most of these compounds, except 12, 13, 19, and 30, were evaluated for their cytotoxicity against five human tumor lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480). In addition, compounds 3, 11, 15, 17, 20, 21, 24, 25, and 29 were tested for their inhibitory activity against NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. This report describes the isolation and structural elucidation of these diterpenoids and the biological activities of selected compounds.

Isodon, including approximately 150 species, is a cosmopolitan and important genus of the Labiatae family.1 Since 1976, more than 60 Isodon species in China have been phytochemically investigated, and more than 1000 new diterpenoids (mainly entkauranoids) have been isolated and characterized by our group.2,3 Some of these compounds exhibit antibacterial, antiinflammatory, and antitumor activities;2 indeed, oridonin,4 eriocalyxin B,5 xerophilusin B,6 and other compounds7 have attracted attention due to their potential application as antitumor agents. Isodon phyllostachys (Diels) Kudo, which is distributed throughout the northwest district of Yunnan Province and the southwest district of Sichuan Province in the People’s Republic of China, has been used as an antiphlogistic and antibiotic agent in folk medicine.8 Previous phytochemical investigations of this species (collected from several places in China) resulted in the isolation of 17 new entkaurane diterpenoids, including two enmein-type ent-kauranoids (phyllostacins A and B),9 one 7,20-cyclo-ent-kauranoid (phyllostachysin A),10 one C-20 oxygenated nonepoxy entkauranoid (phyllostachysin B),11 five 7,20-epoxy-ent-kauranoids (phyllostachysin C, phyllostacins C, D, F, and G),12−14 two 7,20:14,20-diepoxy-ent-kauranoids (phyllostacins H and I),14 one spirolactone-type ent-kauranoid (phyllostacin E),13 and five C-20 nonoxygenated ent-kauranoids (phyllostachysins D−H).15 Among the 17 diterpenoids listed above, only phyllostachysins D, F, G, and H were reported to show significant activities against K562 human cancer cells.9−15 In the course of a systematic search for additional bioactive diterpenoids, the aerial parts of I. phyllostachys (collected in Muli and Yanyuan Counties of Sichuan Province) were © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 6, 2015

A

DOI: 10.1021/acs.jnatprod.5b00802 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

based on the sodium adduct ion at m/z 401.1569 [M + Na]+ (calcd 401.1571) in the HRESIMS and on NMR spectroscopy data (Tables 1 and 2) indicating eight indices of hydrogen deficiency. The IR spectrum showed absorption bands at 3430, 1761, and 1717 cm−1, consistent with the presence of hydroxy, carbonyl, and lactonic carbonyl groups.29 1H NMR data (Table 1) showed resonances that were characteristic of three methyl groups at δH 1.48 (3H, s), 1.12 (3H, s), and 0.83 (3H, s) and of four oxygenated methines at δH 5.93 (1H, s), 5.81 (1H, s), 5.30 (1H, dd, 9.3, 1.3 Hz), and 3.50 (1H, d, 4.2 Hz). 13C NMR and DEPT (Table 2) data revealed 20 carbon signals, including three methyl, four methylene, seven methine (four of which were oxygenated), three quaternary carbons, a lactone carbonyl carbon, a ketocarbonyl carbon, and an oxygenated tertiary carbon. On the basis of the data listed above and the structures of enmein-type ent-kauranoids that were previously isolated from Isodon,2 compound 1 was tentatively assigned as an enmein-type diterpenoid. The structure of 1 was established based on the following 2D NMR correlations and the degree of unsaturation: (a) all protons were assigned to related carbon resonances via HSQC spectroscopic data; (b) 1H−1H COSY data revealed two spin systems, H-1/H2-2/H-3 and H-9/H2-11/H2-12/H-13/H2-14 (Figure 1); (c) HMBC correlations were observed from H-5 (δH 2.23, s) to C-3, C-4, C-9, C-10, C-18, C-19, and C-20, from H-9 (δH 3.06, dd, J = 12.2, 5.0 Hz) to C-1, C-10, C-14, C-15, and C-20, and from H-13 (δH 2.49, dd, J = 10.4, 3.5 Hz) to C-8, C-16, and C-17, indicating that 1 was an enmein-type entkauranoid; (d) HMBC correlations were observed from H-20 (δH 5.81, s) to C-3 and C-6 and from H-5 and H-20 to C-6 (δC 100.3), indicating that 1 possessed a rare 3,20:6,20-diepoxy moiety and a hemiacetal moiety at C-6; (e) HMBC correlations

■

RESULTS AND DISCUSSION Phyllostacin J (1) was obtained as colorless rectangular crystals from MeOH. The molecular formula C20H26O7 was determined

Table 1. 1H NMR Spectroscopic Data for Compounds 1−7 (δ in ppm, J in Hz)a position 1 2a 2b 3a 3b 5 6a 6b 9 11a 11b 12a 12b 13 14a 14b 15 16 17a 17b 18 19 20a 20b MeO -17 a

1 5.30, 2.34, 1.97, 3.50,

dd (9.3, 1.3) overlap overlap br d (4.2)

2 5.22, 2.33, 1.95, 3.46,

d (9.4) overlap m br d (3.9)

3

4

5

6

7

5.01, dd (12.1, 3.6) 1.91, m 1.79, m 1.58, m

4.64, overlap 1.90, overlap 1.83, overlap 1.37, overlap 1.23, overlap 1.77, d (4.7) 3.86, dd (8.9, 4.8) 3.73, d (8.9) 2.74, dd (13.3, 5.7) 1.43, overlap 1.23, overlap 1.71, overlap 1.29, overlap 2.32, overlap 2.56, d (11.8) 1.87, overlap

4.76, overlap 2.81, m 1.86, overlap 1.62, m 1.22, overlap 1.89, overlap 4.17, dd (9.1, 1.8) 3.82, dd (9.1, 7.0) 1.91, overlap 1.47 overlap 1.35, overlap 1.71, overlap 1.30, overlap 2.37, overlap 2.60, d (11.9) 1.96, dd (12.0, 3.5)

5.73, dd (10.2, 7.6) 1.86, m

5.78, dd (10.8, 7.0) 1.84, overlap

1.51, 1.32, 2.77, 5.79,

1.51, 1.33, 2.73, 5.80,

2.35, overlap 1.03, d (6.8)

2.40, overlap 0.98, overlap

0.84, s 0.97, s 6.01, s

0.91, s 1.39, s 5.97, d (4.0)

2.23, br s 5.93, br s

2.28, s 5.95, s

2.41, s 5.54, s

3.06, dd (12.2, 5.0) 2.34, overlap 1.62, overlap 2.02, overlap 1.62, overlap 2.49, m 2.96, dd (11.5, 3.9) 2.55, d (11.5)

3.75, dd (13.3, 5.0) 2.32, overlap 1.52, m 2.14, m 1.69, overlap 2.70, m 2.07, d (11.5) 1.73, overlap 5.53, s

2.95, d (9.1) 4.26, overlap

1.48, s

5.43, 5.12, 0.82, 1.13, 5.80,

6.10, 5.34, 0.99, 0.98, 4.28, 3.67,

0.83, s 1.12, s 5.81, s

s s s s s

2.25, m 2.01, tt (9.7, 4.9) 3.07, br s 2.68, d (12.1) 2.18, dd (12.1, 5.0)

s s s s d (8.2) d (8.2)

m m s s

m m s s

2.89, overlap 5.12, d (3.9)

2.99, d (3.4) 5.16, dd (8.0, 3.9)

2.17, overlap 2.07, dd (15.7, 4.6) 2.89, overlap 3.82, d (11.2) 2.17, overlap

2.47, overlap 1.80, overlap 2.71, overlap 3.63, dd (11.5, 1.3) 2.47, overlap

3.01, m 3.66, dd (10.2, 4.5) 3.46, t (9.8) 0.97, s 0.99, s 4.53, d (8.8) 4.29, d (8.8) 3.11, s

2.42, 3.57, 3.51, 0.96, 0.99, 4.51, 4.29, 3.14,

m m dd (9.5, 4.4) s s d (8.9) d (8.9) s

Recorded at 600 MHz in pyridine-d5. B



Article

Table 2. 13C NMR Spectroscopic Data for Compounds 1−7 (δ in ppm)a

a

position

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 MeO-17

72.0, 32.3, 74.9, 35.4, 49.7, 100.3, 171.2, 55.6, 39.1, 48.9, 19.6, 22.1, 41.0, 31.0, 212.2, 78.1, 19.7, 29.2, 25.1, 99.4,

2 CH CH2 CH C CH CH C C CH C CH2 CH2 CH CH2 C C CH3 CH3 CH3 CH

71.3, 32.9, 74.7, 35.2, 49.7, 100.2, 175.3, 51.6, 31.1, 48.7, 19.0, 32.7, 37.2, 33.3, 78.6, 159.3, 108.3, 29.4, 25.3, 99.5,

3 CH CH2 CH C CH CH C C CH C CH2 CH2 CH CH2 CH C CH2 CH3 CH3 CH

76.9, 23.8, 40.6, 30.6, 53.6, 99.7, 170.2, 58.3, 48.8, 45.6, 63.1, 40.3, 36.2, 35.3, 198.6, 146.2, 118.2, 34.0, 24.4, 74.6,

4 CH CH22 CH2 C CH CH C C CH C CH CH2 CH CH2 C C CH2 CH3 CH3 CH2

76.0, 23.7, 37.3, 31.7, 49.5, 66.4, 172.6, 55.9, 37.0, 51.0, 18.2, 18.9, 31.9, 34.6, 212.6, 49.8, 10.4, 32.6, 23.1, 99.4,

5 CH CH2 CH2 C CH CH2 C C CH C CH2 CH2 CH CH2 C CH CH3 CH3 CH3 CH

76.7, 24.9, 36.8, 31.9, 47.8, 68.4, 172.3, 56.7, 44.5, 53.1, 18.3, 19.1, 32.6, 34.2, 215.3, 49.3, 10.3, 33.6, 23.0, 103.1,

6 CH CH2 CH2 C CH CH2 C C CH C CH2 CH2 CH CH2 C CH CH3 CH3 CH3 CH

78.7, 23.9, 37.3, 31.4, 55.6, 101.9, 172.2, 57.3, 48.3, 49.7, 65.3, 31.6, 31.0, 35.5, 212.6, 54.9, 69.4, 33.0, 23.2, 74.1, 58.4,

7 CH CH2 CH2 C CH CH C C CH C CH CH2 CH CH2 C CH CH2 CH3 CH3 CH2 CH3

78.2, 23.9, 37.3, 31.5, 55.6, 101.9, 171.8, 57.0, 48.2, 49.7, 65.9, 41.1, 31.7, 34.3, 212.3, 56.9, 71.7, 33.0, 23.2, 73.9, 58.5,

CH CH2 CH2 C CH CH C C CH C CH CH2 CH CH2 C CH CH2 CH3 CH3 CH2 CH3

Recorded at 150 MHz in pyridine-d5.

Figure 1. 1H−1H COSY (bold), selected HMBC (arrow), and key ROESY correlations of 1.

were observed from H-12 (δH 2.02), H-13, H-14 (δH 2.55, d, J = 11.5 Hz), and Me-17 (δH 1.48, s) to C-16 (δC 78.1), implying the presence of an OH group at C-16; (f) ROESY correlations observed for Me-17/H-12α, H-20/H-2α, H-9/H-20, and H-6/ Me-19α demonstrated that Me-17, C-20, H-9, and H-6 were αoriented, respectively; (g) ROESY correlations observed between H-1, H-3, and H-5 and Me-18β showed that H-1, H-3, and H-5 were β-oriented, respectively; and (h) ROESY correlation of H-14β/H-1 suggested that C-14 adopted a βorientation (Figure 1). A single crystal of 1 was obtained from MeOH and subjected to X-ray diffraction analysis. The final refinement based on Cu Kα data resulted in a Flack parameter30 of 0.23(15) and a Hooft parameter31 of 0.12(5) for 1300 Bijvoet pairs, enabling the absolute configuration of 1 to be assigned unambiguously as (1S, 3R, 5R, 6R, 8S, 9S, 10R, 13R, 16S, 20R) (Figure 2). Thus, the structure of 1 was defined as 3α,20:6,20-diepoxy-6β,16βdihydroxy-6,7-seco-ent-kaur-15-on-1α,7-olide and given the trivial name phyllostacin J. Phyllostacin K (2) was determined to have the molecular formula C20H26O6 based on HRESIMS and 13C NMR data, corresponding to eight indices of hydrogen deficiency. IR absorption bands at 3434, 1718, and 1632 cm−1 indicated the presence of hydroxy, lactone carbonyl, and double-bond functionalities. On the basis of NMR data (Tables 1 and 2), compound 2 possessed an enmein-type ent-kauranoid skeleton

Figure 2. ORTEP drawing of compound 1.

similar to that of compound 1. Olefinic carbon resonances indicated the presence of an exocyclic Δ16,17 carbon−carbon double bond in 2 instead of the oxygenated tertiary carbon (δC 78.1) and a methyl carbon (δC 19.7) that occurred in 1. In addition, a carbonyl carbon (δC 212.2) that occurred in 1 was replaced by an oxygenated methine carbon (C-15, δC 78.6) in 2. HMBC correlations from H-9 (δH 3.75, dd, J = 13.3, 5.0 Hz), H-13 (δH 2.70, m), H-14β (δH 2.07, d, J = 11.5 Hz), and H2-17 (δH 5.43, s and 5.12, s) to C-15 implied the presence of an OH group at C-15. HO-15 was assigned to be α-oriented due to the shielded C-9 (δC 31.1), which is caused by the γ-steric compression effect between HO-15 and H-9α; this orientation was supported by the ROESY correlation between H-15 (δH 5.53, s) and H-14α (δH 1.73). Comparison of the ROESY spectrum of 2 with that of 1 indicated that the relative configurations of the stereogenic carbons in 2 were identical to those in 1. Accordingly, compound 2 was identified as 3α,20:6,20-diepoxy-6β,15α-dihydroxy-6,7-seco-ent-kaur-16-en1α,7-olide and given the trivial name phyllostacin K. Phyllostacin L (3) was isolated as a white, amorphous powder, and its molecular formula was established as C20H24O5 C



Article

Table 3. 1H NMR Spectroscopic Data for Compounds 8−13 (δ in ppm, J in Hz) 8b

position 1 2a 2b 3a 3b 5 6 9 11a 11b 12a 12b 13 14a 14b 15 16 17a 17b 18 19a 19b 20a 20b MeO-20 a

4.65, m 1.87, overlap 1.38, 1.28, 2.22, 5.56, 3.19, 1.74, 1.38, 2.07, 1.50, 2.30, 2.56, 1.88,

overlap m s s dd (12.8, 5.5) m overlap m m overlap d (11.9) overlap

9b 4.60, 2.40, 1.78, 1.45, 1.22, 2.14, 5.87, 2.40, 1.71, 1.37, 2.06, 1.44, 2.40, 2.60, 1.97,

dd (11.4, 5.7) overlap overlap overlap overlap s s overlap m m m overlap overlap d (11.9) d (11.9)

10a

11b

4.61, t (8.6) 1.83, m 1.34, 1.21, 2.13, 5.72, 2.93, 2.12, 1.56, 2.11, 1.28, 1.96, 2.44, 2.15,

m m s d (2.7) m overlap m overlap m br s d (12.1) overlap

2.30, overlap 0.96, d (6.7)

2.41, overlap 0.93, d (5.4)

2.02, q (7.6) 1.04, d (7.7)

0.98, s 1.00, s

1.05, s 1.19, s

0.99, s 0.94, s

5.42, s

5.44, s

4.40, m 4.22, d (8.9)

3.63, s

3.35, s

12a

13a

4.64, 2.43, 1.82, 1.46, 1.21, 2.16, 5.89, 2.68, 2.16, 1.60, 2.16, 1.35, 2.88, 2.56, 2.05,

dd (11.4, 5.9) m m m overlap s d (3.0) dd (13.0, 4.8) overlap m overlap m m d (11.8) dd (11.8, 4.4)

4.76, 2.01, 1.89, 1.61, 1.41, 2.57, 5.94, 2.92, 1.54, 1.58, 2.01, 1.69, 2.67, 2.14, 1.49, 5.59,

dd (11.9, 4.8) overlap overlap overlap overlap d (5.8) d (5.8) dd (12.7, 5.5) overlap overlap overlap overlap overlap overlap overlap s

4.71, 3.15, 1.85, 1.71, 1.46, 2.50, 6.21, 2.63, 1.42, 1.43, 2.00, 1.68, 2.67, 2.14, 1.49, 5.50,

dd (11.9, 5.2) m overlap overlap overlap d (5.8) overlap dd (13.2, 5.4) overlap overlap overlap overlap overlap overlap overlap s

5.94, 5.25, 1.06, 1.20,

s s s s

5.44, 5.21, 1.02, 3.85, 3.40, 6.14,

s s s d (8.9) overlap d (9.4)

5.46, 5.19, 1.10, 4.92, 3.40, 6.20,

s s s d (7.6) overlap overlap

5.47, s 3.38, s

Recorded at 600 MHz in pyridine-d5. bRecorded at 400 MHz in pyridine-d5.

based on HRESIMS and 13C NMR data, showing nine indices of hydrogen deficiency. 13C NMR and DEPT data (Table 2) of 3 exhibited 20 resonances representing two methyl, six methylene (one oxygenated and one olefinic), six methine (three oxygenated), and four quaternary carbons (one olefinic), a conjugated carbonyl carbon, and a lactone carbonyl carbon. Comparisons of the 1H and 13C NMR data of 3 with those of nodosin (17)19 indicated that both compounds had similar skeletons and substitution patterns. The only difference was that an epoxy link was present between C-6 and C-11 in 3. This conclusion was verified by the HMBC correlation between H-6 (δH 5.54, s) and C-11 (δC 63.1) and by the nine indices of hydrogen deficiency in 3 rather than the eight indices in 17. The ROESY correlations of H-6/Me-19α (δH 0.98, s) and H-11 (δH 4.26)/H-20α (δH 3.67, d, J = 8.2 Hz) indicated that H-6 and H-11 were α-oriented in 3. Therefore, the structure of compound 3 was established as 6β,11β:6,20-diepoxy-6,7-secoent-kaur-16-en-15-on-1α,7-olide and given the trivial name phyllostacin L. Phyllostacin M (4) and 20-epiphyllostacin M (5) were obtained as a pair of epimers. The molecular formula of these compounds was C20H28O5 according to HRESIMS and 13C NMR data. The 1H and 13C NMR data of these compounds (Tables 1 and 2) were similar to those of phyllostacin B (23).14 The difference between the compounds was that the C-6 hemiacetal moiety in 23 was replaced by an oxygenated methylene in 4 and 5. This assignment was confirmed by (a) 1 H−1H COSY cross-peaks between H2-6 and H-5; (b) HMBC correlations from H2-6 (δH 3.86, dd, J = 8.9, 4.8 Hz and 3.73, d, J = 8.9 Hz) to C-4, C-5, C-10, and C-20 in 4; and (c) HMBC correlations from H2-6 (δH 4.17, dd, J = 9.1, 1.8 Hz and 3.82, dd, J = 9.1, 7.0 Hz) to C-4, C-5, C-10, and C-20 in 5. The ROESY correlations of H-20 (δH 6.01, s)/Me-19α (δH 0.97, s)

in 4 and of H-20 (δH 5.97, d, J = 4.0 Hz)/H-9α (δH 1.91) in 5 demonstrated that H-20 in 4 was β-oriented and that H-20 in 5 was α-oriented. This conclusion was further supported by the observation of shielding of C-9 (δC 37.0, Δ 7.5 ppm) due to γsteric compression between HO-20α and H-9α in 4. The ROESY correlation of Me-17/H-12α suggested that Me-17 adopted an α-orientation; this suggestion was supported by the ROESY correlation between H-16 and H-14α. Thus, the structures of compounds 4 and 5, named phyllostacin M and 20-epiphyllostacin M, were identified as 6,20-epoxy-20αhydroxy-16α-methyl-6,7-seco-ent-kaur-15-on-1α,7-olide (4) and 6,20-epoxy-20β-hydroxy-16α-methyl-6,7-seco-ent-kaur-15-on1α,7-olide (5), respectively. The molecular formula of 11-epitaibaijaponicain A (6) was C21H30O7 according to HRESIMS and 13C NMR data. The 13C NMR data of 6 resembled those of taibaijaponicain A,32 except for some differences regarding the chemical shifts of C-9 (δC 48.3 in 6 and 53.1 in taibaijaponicain A), C-11 (δC 65.3 in 6 and 63.3 in taibaijaponicain A), and C-16 (δC 54.9 in 6 and 58.8 in taibaijaponicain A). Compound 6 differed from taibaijaponicain A only in the orientation of HO-11, which was deduced from the 1H NMR and ROESY spectra of 6. The ROESY correlations of HO-11 (δH 6.80) with H-1β (δH 5.73, dd, J = 10.2, 7.6 Hz), H-5β (δH 2.77, s), and H-14β (δH 3.82, d, J = 11.2 Hz) indicated that HO-11 adopted a β-orientation in 6. The structure of 6 was thus assigned as 6,20-epoxy-6β,11βdihydroxy-16α-methoxymethyl-6,7-seco-ent-kaur-15-on-1α,7olide and given the trivial name 11-epitaibaijaponicain A. 11,17-Diepitaibaijaponicain A (7) had the same molecular formula, C21H30O7, as compound 6 according to HRESIMS data (m/z 417.1887 [M + Na]+). The 13C NMR data (Table 2) of 7 were similar to those of 6 except those of C-17. The ROESY correlations of H-16 (δH 2.42, m)/H-12α (δH 1.80) D



Article

Table 4. 13C NMR Spectroscopic Data for Compounds 8−13 (δ in ppm) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 MeO-20 a

8b 76.1, 23.9, 37.6, 31.3, 55.0, 99.5, 172.8, 56.3, 39.2, 51.1, 19.3, 20.4, 32.1, 35.0, 212.0, 49.5, 11.0, 32.8, 23.6, 108.8, 57.0,

CH CH2 CH2 C CH CH C C CH C CH2 CH2 CH CH2 C CH CH3 CH3 CH3 CH CH3

9b 76.7, 24.9, 37.7, 31.2, 54.1, 101.6, 172.3, 57.3, 45.5, 52.3, 18.8, 19.3, 32.8, 34.7, 215.6, 49.4, 10.6, 33.9, 22.7, 111.6, 55.5,

CH CH2 CH2 C CH CH C C CH C CH2 CH2 CH CH2 C CH CH3 CH3 CH3 CH CH3

10a 76.5, 23.7, 37.2, 31.0, 54.4, 101.9, 172.0, 57.2, 45.8, 50.0, 19.9, 29.0, 35.5, 32.3, 214.9, 51.0, 16.0, 32.7, 23.0, 73.9,

11b

CH CH2 CH2 C CH CH C C CH C CH2 CH2 CH CH2 C CH CH3 CH3 CH3 CH2

76.5, 25.0, 37.6, 31.2, 54.3, 101.6, 171.6, 56.9, 44.9, 52.6, 19.3, 29.7, 35.2, 33.0, 200.9, 151.3, 117.8, 33.8, 22.6, 111.4, 55.5

CH CH2 CH2 C CH CH C C CH C CH2 CH2 CH CH2 C C CH2 CH3 CH3 CH

12a 75.5, 23.4, 29.2, 40.9, 52.9, 104.8, 175.1, 52.2, 30.1, 49.7, 18.9, 33.5, 36.8, 32.0, 78.0, 159.0, 108.5, 30.4, 76.2, 100.4,

CH CH2 CH2 C CH CH C C CH C CH2 CH2 CH CH2 CH C CH2 CH3 CH2 CH

13a 76.4, 24.6, 29.4, 41.0, 52.7, 111.4, 175.6, 52.8, 36.4, 52.1, 18.4, 33.4, 36.8, 32.6, 78.0, 159.6, 108.2, 30.9, 74.6, 102.4,

CH CH2 CH2 C CH CH C C CH C CH2 CH2 CH CH2 CH C CH2 CH3 CH2 CH

Recorded at 150 MHz in pyridine-d5. bRecorded at 100 MHz in pyridine-d5.

and H2-17 (δH 3.57, m and 3.51, dd, J = 9.5, 4.4 Hz)/H-14α (δH 2.47) indicated that H-16 was α-oriented and C-17 was βoriented in 7. Therefore, the structure of compound 7 was defined as 6,20-epoxy-6β,11β-dihydroxy-16β-methoxymethyl6,7-seco-ent-kaur-15-on-1α,7-olide and given the trivial name 11,17-diepitaibaijaponicain A. Phyllostacin N (8) was obtained as colorless needles from MeOH. On the basis of HRESIMS and 13C NMR data, the molecular formula of this compound was C21H30O6. The 13C NMR and DEPT data (Table 4) of 8 exhibited 21 resonances representing four methyl (one oxygenated), five methylene, seven methine (three oxygenated), and three quaternary carbons, a carbonyl carbon, and a lactone carbonyl carbon. The spectroscopic data indicated that the structure of 8 was similar to that of dihydroisodocarpin (16)18 except that an oxygenated methine (C-20, δC 108.8) was present in 8 rather than the oxygenated methylene (δC 74.1) in 16 and the presence of a methoxy group (δC 57.0) in 8. The HMBC correlation between OMe (δH 3.63, s) and C-20 showed that the OMe group was linked to C-20. In the ROESY data, H-20 (δH 5.42, s) was correlated with Me-19α (δH 1.00, s) and H-2α (δH 1.87), suggesting that H-20 was β-oriented. This conclusion was further supported by the observed shielding of C-9 (δC 39.2, Δ 5.7 ppm), which was caused by γ-steric compression between OMe-20α and H-9α. The ROESY correlations of Me-17 (δH 0.96, d, J = 6.7 Hz)/H-11α (δH 1.38) and H-16 (δH 2.30)/H-14α (δH 1.88) indicated that Me17 was α-oriented and H-16 was β-oriented. To determine the absolute configuration of 8, single-crystal X-ray diffraction was performed. The Hooft parameter [0.11(7)] for 1347 Bijvoet pairs and the Flack parameter [0.2(2)] verified the αorientation of OMe at C-20, and the stereogenic centers of 8 were assigned the following absolute configurations: 1S, 5R, 6R, 8S, 9S, 10R, 13R, 16R, 20S (Figure 4). Thus, the structure of compound 8 was assigned as 6,20-epoxy-6β-hydroxy-20αmethoxy-16α-methyl-6,7-seco-ent-kaur-15-on-1α,7-olide and given the trivial name phyllostacin N.

Figure 3. 1H−1H COSY (bold), selected HMBC (arrow), and key ROESY correlations of 8.


20-Epiphyllostacin N (9) was isolated as colorless needles from MeOH and had the same molecular formula as 8. The 13C NMR data (Table 4) resembled those of 8 except for small differences in the chemical shifts of C-2, C-6, C-8, C-9, C-15, and C-20. Compound 9 differed from 8 only in the orientation of MeO-20, as deduced from the key ROESY correlation of H20 (δH 5.44, s)/H-9α (δH 2.40) in 9, which indicated that H-20 was α-oriented. Single-crystal X-ray diffraction analysis using the anomalous scattering of Cu Kα radiation with a Flack E



Article

5 (δC 52.7), C-19 (δC 74.6), and C-20 (δC 102.4) in 13. The ROESY correlations of H-20 (δH 6.14, d, J = 9.4 Hz)/H-19α (δH 3.85, d, J = 8.9 Hz) in 12 and H-20 (δH 6.20)/H-9α (δH 2.63, dd, J = 13.2, 5.4 Hz) in 13 demonstrated that H-20 was βoriented in 12 and α-oriented in 13. These findings were further supported by the γ-steric compression between HO-20α and H-9α in 12, which caused shielding of C-9 (δC 30.1, Δ 6.3 ppm). HO-15 was assigned an α-orientation due to the shielded C-9, which was caused by γ-steric compression between HO-15 and H-9α. The ROESY correlation H-6/H-5β and the coupling constant of H-5 (δH 2.57, d, J = 5.8 Hz in 12, δH 2.50, d, J = 5.8 Hz in 13) suggested that H-6 was β-oriented. Therefore, compounds 12 and 13, named phyllostacin O and 20epiphyllostacin O, respectively, were defined as 6,19:6,20diepoxy-15α,20α-dihydroxy-6,7-seco-ent-kaur-16-en-1α,7-olide (12) and 6,19:6,20-diepoxy-15α,20β-dihydroxy-6,7-seco-entkaur-16-en-1α,7-olide (13), respectively. All isolates except 12, 13, 19, and 30 (due to sample limitations) were evaluated for their in vitro cytotoxicity against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480) using the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method; cis-platin was used as the positive control. Compounds 11, 15, 20, and 21 exhibited moderate cytotoxic activity (IC50 values ranging from 1.2 to 5.0 μM for all tested cell lines), whereas compounds 3, 17, 24, 25, and 29 exhibited modest cytotoxic potency (Table 5). The nine cytotoxic

parameter of 0.9(7) further confirmed the relative configuration of 9 (Figure 5). Therefore, the structure of 9 was defined as 6,20-epoxy-6β-hydroxy-20β-methoxy-16α-methyl-6,7-seco-entkaur-15-on-1α,7-olide and given the trivial name 20-epiphyllostacin N.


According to HRESIMS and 13C NMR data, 17-epidihydroisodocarpin (10) had the molecular formula C20H28O5. The 1 H and13C NMR data (Tables 3 and 4) of 10 were similar to those of dihydroisodocarpin (16),18 except for the chemical shift of Me-17 (δC 16.0 in 10; δC 10.5 in 16). The orientation of Me-17 in 10 differed from that in 16, as confirmed by the ROESY data of 10. The ROESY correlations of H-16 (δH 2.02, q, J = 7.6 Hz)/H-12α (δH 1.28, m) and Me-17 (δH 1.04, d, J = 7.7 Hz)/H-14α (δH 2.15) suggested that H-16 was α-oriented and Me-17 was β-oriented. Thus, compound 10 was identified as 6,20-epoxy-6β-hydroxy-16β-methyl-6,7-seco-ent-kaur-15-on1α,7-olide and given the trivial name 17-epidihydroisodocarpin. 20-Episerrin C (11) had the same molecular formula, C21H28O6, as serrin C (15),17 as deduced from HRESIMS and 13C NMR data. Comparisons of the 1H and 13C NMR data of 11 (Tables 3 and 4) with those of 15 indicated that both compounds had the same skeletons and substitution patterns and differed in the orientation of the OMe group at C-20. This finding was confirmed based on the ROESY correlation of H-20 (δH 5.47, s)/H-9α (δH 2.68, dd, J = 13.0, 4.8 Hz) in 11, indicating that H-20 was α-oriented and MeO-20 was βoriented. Therefore, the structure of 11, named 20-episerrin C, was identified as 6,20-epoxy-6β-hydroxy-20β-methoxy-6,7-secoent-kaur-16-en-15-on-1α,7-olide. The molecular formulas of phyllostacin O (12) and 20epiphyllostacin O (13) were determined as C20H26O6 based on HRESIMS and 13C NMR data indicative of eight indices of hydrogen deficiency. These compounds were isolated as a pair of epimers. The 13C NMR and DEPT data of 12 and 13 exhibited 20 pairs of carbon resonances, including one methyl, seven methylene (one oxygenated and one olefinic), seven methine (four oxygenated), and four quaternary carbons (one olefinic) and a lactone carbonyl carbon. The 1H and 13C NMR data of 12 and 13 (Tables 3 and 4) were similar to those of 14. The most notable difference between the structures was that Me-19 in 14 is replaced by an oxygenated methylene in 12 and 13. This assignment was confirmed by the HMBC correlations from H-6 (δH 5.94, d, J = 5.8 Hz) to C-5 (δC 52.9), C-19 (δC 76.2), and C-20 (δC 100.4) in 12 and from H-6 (δH 6.21) to C-

Table 5. Cytotoxic Activities of Enmein-Type entKauranoids from I. phyllostachys against Five Tumor Cell Linesa compound

HL-60

SMMC-7721

A-549

MCF-7

SW-480

3 11 15 17 20 21 24 25 29 DDPb

15.4 3.5 3.2 10.3 2.1 3.0 16.8 9.2 14.3 3.2

15.8 4.7 5.0 11.4 3.3 4.2 31.8 9.8 12.5 5.8

14.0 1.2 3.4 14.3 2.1 3.9 39.0 13.3 10.2 7.1

14.0 3.9 3.2 14.0 3.4 2.4 >40 15.7 13.7 15.0

15.2 3.9 4.6 10.9 1.7 3.2 14.8 5.5 >40 11.3

a Results are expressed as IC50 values in μM. Cell lines: HL-60, acute leukemia; SMMC-7721, hepatic cancer; A-549, lung cancer; MCF-7, breast cancer; SW-480, colon cancer. Compounds 1, 2, 4−10, 14, 16, 18, 22, 23, 26−28, 31, and 32 were inactive (IC50 > 40 μM) for all cell lines. bDDP (cisplatin) was used as a positive control.

compounds contained an α-exomethylene-cyclopentanone motif. The remaining 17 compounds (1, 2, 4−10, 14, 16, 18, 22, 23, 26, 28, and 32), which lacked this structural motif, were noncytotoxic against the tested cell lines. This result indicates that the α,β-unsaturated carbonyl function is structurally required for cytotoxicity.2 Compounds 27 and 31 also contained this motif but did not exhibit cytotoxicity, possibly due to the hydroxy group substitutions, either at C-3 and C-11 or at C-11 and C-19. These results are consistent with the conclusions of previous structure−activity studies.2,33 Inflammation is part of the complex biological response of body tissues to harmful stimuli that aims to decrease the toxicity of harmful agents and repair damaged tissue. A key feature of the inflammatory response is the activation of F



Article

gel CC; four fractions were eluted with MeOH/H2O (30:70 to 70:30 gradient): C1−C4 (30:70, 40:60, 50:50, and 70:30). Fractions C1 (2 g) and C2 (18 g) were separated into 10 (C1-1− C1-10) and 11 (C2-1−C2-11) subtractions, respectively, using silica gel CC (petroleum ether/acetone gradient, 90:10 to 0:100). C1-4 was purified by silica gel CC (CH3Cl/acetone gradient, 90:10−0:100) to yield 27 (5 mg) and 32 (21 mg). Compounds 23 (20 mg) and 31 (4 mg) were isolated from C1-6 by silica gel CC (CHCl3/acetone gradient, 90:10−0:100) and then by semipreparative HPLC (RP-18, 30% MeCN/H2O). Compound 25 (523 mg) was crystallized from C1-7, and part of the mother liquid of 25 was purified by semipreparative HPLC (RP-18, 40% MeOH/H2O), affording 18 (7 mg) and 26 (5 mg). C2-7 was purified by silica gel CC (CHCl3/ acetone gradient, 90:10−50:50), followed by semipreparative HPLC (RP-18, 31% MeCN/H2O), to yield compounds 24 (6 mg) and 28 (5 mg). Compounds 12 and 13 (2 mg) were obtained from C2-9 by semipreparative HPLC (Cholester, 31% MeCN/H2O). Fractions C3 (16 g) and C4 (9 g) were chromatographed over silica gel and eluted with petroleum ether/acetone (90:10 to 50:50 gradient) to provide 10 (C3-1−C3-10) and seven (C4-1−C4-7) subfractions, respectively. Compound 22 (2 g) was crystallized from C3-3, and part of the residue was resolved using semipreparative HPLC (RP-18, 35% MeCN/H2O) to yield compounds 3 (3 mg), 9 (26 mg), 11 (7 mg), and 21 (19 mg). C3-4 was separated by semipreparative HPLC (RP18, 33% MeCN/H2O) to yield compounds 8 (144 mg), 15 (13 mg), and 19 (1 mg). C3-5 was purified by silica gel CC (CHCl3/acetone gradient, 95:5−50:50), followed by semipreparative HPLC (Cholester, 46% MeOH/H2O), to obtain a mixture of compounds 4 and 5 (36 mg). Compounds 1 (13 mg), 2 (7 mg), 6 (5 mg), and 7 (8 mg) were isolated from C3-7 following the same method as described for C3-5. Compound 17 (3 g) crystallized from C3-8, and the residue was separated by semipreparative HPLC to afford compound 14 (19 mg). C4-5 was separated by silica gel CC (CHCl3/acetone gradient, 95:5− 70:30), followed by semipreparative HPLC (RP-18, 35% MeCN/ H2O), to afford compounds 20 (31 mg), 29 (50 mg), and 30 (1 mg). Semipreparative HPLC (Cholester, 35% MeCN/H2O) of C4-6 afforded compounds 10 (33 mg) and 16 (5 mg). Phyllostacin J (1): colorless, rectangular crystals; mp 219−220 °C; [α]23D −108 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 205 (3.0) nm; IR (KBr) νmax 3430, 2961, 1761, 1717, 1279, 1259, 1112, 1050, 985 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive HRESIMS [M + Na]+ m/z 401.1569 (calcd for C20H26O7Na, 401.1571). Phyllostacin K (2): white, amorphous powder; [α]23D −103 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 203 (3.8) nm; IR (KBr) νmax 3434, 2960, 2934, 1718, 1632, 1239, 1129, 1057, 1046 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive HRESIMS [M + Na ]+ m/z 385.1624 (calcd for C20H26O6Na, 385.1622). Phyllostacin L (3): white, amorphous powder; [α]23D −76 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 227 (3.4), 202 (3.4) nm; IR (KBr) νmax 3440, 2955, 2930, 1764, 1722, 1632, 1260, 1070 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive HRESIMS [M + Na]+ m/z 367.1516 (calcd for C20H24O5Na, 367.1516). Phyllostacin M (4) and 20-Epiphyllostacin M (5): white, amorphous powder; [α]23D −114 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 203 (2.7) nm; IR (KBr) νmax 3439, 2953, 1750, 1716, 1631, 1273, 1142, 1051 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive HRESIMS [M + Na]+ m/z 371.1832 (calcd for C20H28O5Na, 371.1829). 11-Epitaibaijaponicain A (6): white, amorphous powder; [α]23D −170 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (3.1) nm; IR (KBr) νmax 3475, 2940, 1751, 1712, 1251, 1050, 1021 cm−1; 1H and 13 C NMR data, see Tables 1 and 2; positive HRESIMS [M + Na]+ m/z 417.1884 (calcd for C21H30O7Na, 417.1884). 11,17-Diepitaibaijaponicain A (7): white, amorphous powder; [α]23D −126 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203 (3.0) nm; IR (KBr) νmax 3522, 3438, 2932, 1752, 1703, 1632, 1253, 1047 cm−1; 1 H and 13C NMR data, see Tables 1 and 2; positive HRESIMS [M + Na]+ m/z 417.1887 (calcd for C21H30O7Na, 417.1884). Phyllostacin N (8): colorless needles (MeOH); mp 180−181 °C; [α]23D −163 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 201 (3.1) nm;

phagocytic cells that are involved in the host defense. Nitric oxide (NO), which is produced by the inducible NO synthase (iNOS) isoform, is an essential component of the host innate immune and inflammatory response to a variety of pathogens.34 Owing to the aforementioned structure−activity conclusions, nine compounds (3, 11, 15, 17, 20, 21, 24, 25, 29) containing an α-exomethylene-cyclopentanone motif and exhibiting cytotoxic activity were tested for their inhibitory activity against NO production in LPS-stimulated RAW264.7 cells using the MTT assay. All compounds (except 24) exhibited inhibitory effects (IC50 values ranging from 0.74 to 4.93 μM). Compound 24 exhibited moderate activity (IC50: 7.42 μM) (Table 6). Table 6. Inhibitory Effects of Enmein-Type ent-Kauranoids from I. phyllostachys on LPS-Activated NO Production in RAW264.7 Cells

a

compound

IC50 (μM)

compound

IC50 (μM)

3 11 15 17 20

4.93 1.36 1.56 3.56 0.74

21 24 25 29 MG-132a

1.47 7.42 3.53 3.51 0.15

Positive control.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were obtained on an XRC-1 apparatus and were uncorrected. Optical rotations were measured on Horiba SEPA-300 and JASCO P-1020 polarimeters. UV spectra were recorded on a Shimadzu UV-2401A spectrophotometer. A Tenor 27 FT-IR spectrometer was used for scanning IR spectroscopy using KBr pellets. NMR spectra were recorded on Bruker AM-400, DRX-500, and DRX-600 spectrometers with TMS as internal standard. HRESIMS data were acquired on an Agilent 6540 QSTAR TOF time-of-flight mass spectrometer. X-ray data were collected on a Bruker APEX DUO diffractometer using Cu Kα radiation. Analytical and semipreparative HPLC was performed on an Agilent 1260 or 1100 apparatus with a Zorbax SB-C18 (Agilent, 4.6 mm × 250 mm, 9.4 mm × 250 mm) column or COSMOSIL Cholester Packed column (Nacalai, 4.6 i.d. × 250 mm, 10 i.d. × 250 mm,). Column chromatography (CC) was performed on silica gel (100−200 mesh and 200−300 mesh; Qingdao Marine Chemical, Inc., Qingdao, People’s Republic of China), Lichroprep RP-18 gel (40−63 μm, Merck, Darmstadt, Germany), and MCI gel (75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan). Thin-layer chromatography (TLC) was carried out on silica gel 60 F254 on glass plate (Qingdao Marine Chemical, Inc.), and spots were visualized by UV light (254 nm) and sprayed with 10% H2SO4 in ethanol, followed by heating. Plant Material. The aerial parts of I. phyllostachys were collected in August 2011 from two areas (Muli and Yanyuan) of Sichuan Province, People’s Republic of China, and identified by Prof. Xi-Wen Li at the Kunming Institute of Botany. A voucher specimen (KIB20110822) has been deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences. Extraction and Isolation. The air-dried and powered aerial parts of I. phyllostachys (11.0 kg) were extracted with 70% aqueous acetone (5 × 35 L, 2 days each) at room temperature and then filtered. The filtrate was evaporated in vacuo to afford a residue (approximately 15 L), which was partitioned by liquid−liquid extraction between EtOAc and H2O. The EtOAc extract (765 g) was chromatographed over silica gel (4 kg, 100−200 mesh) and eluted with CHCl3/acetone (100:0− 0:100 gradient) to afford fractions A−G. Fraction C (CHCl3/acetone, 80:20; 120 g) was decolorized on MCI and eluted with 90:10 MeOH/ H2O to yield a yellowish-white gum (96 g). The part (47 g) of the gum that was readily soluble in MeOH was subjected to RP-18 silica G



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IR (KBr) νmax 3500, 2952, 1760, 1714, 1452, 1233, 1131, 986 cm−1; 1 H and 13C NMR data, see Tables 3 and 4; positive HRESIMS [M + Na]+ m/z 401.1937 (calcd for C21H30O6Na, 401.1935). 20-Epiphyllostacin N (9): colorless needles (MeOH); mp 189−190 °C; [α]23D −180 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 201 (2.9) nm; IR (KBr) νmax 3458, 2950, 1754, 1702, 1260, 1040, 990 cm−1; 1H and 13C NMR data, see Tables 3 and 4; positive HRESIMS [M + Na]+ m/z 401.1937 (calcd for C21H30O6Na, 401.1935). 17-Epidihydroisodocarpin (10): white, amorphous powder; [α]23D −136 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 205 (2.9) nm; IR (KBr) νmax 3454, 2932, 1756, 1703, 1632, 1258, 1055 cm−1; 1H and 13 C NMR data, see Tables 3 and 4; positive HRESIMS [M + Na]+ m/z 371.1831 (calcd for C20H28O5Na, 371.1829). 20-Episerrin C (11): white, amorphous powder; [α]23D −179 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 232 (3.8) nm; IR (KBr) νmax 3440, 2951, 1753, 1711, 1642, 1258, 1145, 1048, 994 cm−1; 1H and 13C NMR data, see Tables 3 and 4; positive HRESIMS [M + Na]+ m/z 399.1780 (calcd for C21H28O6Na, 399.1778). Phyllostacins O (12) and 20-Epiphyllostacin O (13): white, amorphous powder; [α]23D −114 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 203 (3.7) nm; IR (KBr) νmax 3430, 2931, 1730, 1632, 1239, 1059 cm−1; 1H and 13C NMR data, see Tables 3 and 4; positive HRESIMS [M + Na]+ m/z 385.1623 (calcd for C20H26O6Na, 385.1622). X-ray Crystal Structure Analysis. Crystals of 1, 8, and 9 were obtained in MeOH. The intensity data for phyllostacin J (1), phyllostacin N (8), and 20-epiphyllostacin N (9) were collected at 100 K on a Bruker APEX DUO diffractometer equipped with an APEX II CCD using Cu Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT. The structures were solved by direct methods using SHELXS-97,35 expanded using difference Founier techniques, and refined by the program and full-matrix least-squares calculations. The non-hydrogen atoms were refined anisotropically, and hydrogen atoms were fixed at calculated positions. Crystallographic data (excluding structure factor tables) for the reported structures have been deposited with the Cambridge Crystallographic Data Center (CCDC) as supplementary publications no. CCDC 1406789 for 1, CCDC 1406790 for 8, and CCDC 1406791 for 9. Copies of the data can be obtained free of charge from the CCDC, 12 Union Road, Cambridge, CB 1EZ, UK [fax: int. + 44(0) (1223) 336 033); e-mail: [email protected]]. Crystallographic data for phyllostacin J (1): C20H26O7·H2O, Mw = 396.42, monoclinic, a = 7.43150(10) Å, b = 9.3881(2) Å, c = 13.1958(3) Å, α = 90.00°, β = 91.3240(10)°, γ = 90.00°, V = 920.39(3) Å3, T = 100(2) K, space group P21, Z = 2, μ(Cu Kα) = 0.923 mm−1, 7775 reflections measured, 3099 independent reflections (Rint = 0.0376). The final R1 values were 0.0403 (I > 2σ(I)). The final wR(F2) values were 0.1064 (I > 2σ(I)). The final R1 values were 0.0403 (all data). The final wR(F2) values were 0.1064 (all data). The goodness of fit on F2 was 1.067. Flack parameter = 0.23(15). The Hooft parameter is 0.12(5) for 1300 Bijvoet pairs. Crystallographic data for phyllostacin N (8): C21H30O6, M = 378.45, orthorhombic, a = 15.0447(4) Å, b = 19.0079(5) Å, c = 6.6069(2) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1889.36(9) Å3, T = 100(2) K, space group P21212, Z = 4, μ(Cu Kα) = 0.790 mm−1, 9953 reflections measured, 3304 independent reflections (Rint = 0.0417). The final R1 values were 0.0633 (I > 2σ(I)). The final wR(F2) values were 0.1891 (I > 2σ(I)). The final R1 values were 0.0637 (all data). The final wR(F2) values were 0.1899 (all data). The goodness of fit on F2 was 1.075. Flack parameter = 0.2(2). The Hooft parameter is 0.11(7) for 1347 Bijvoet pairs. Crystallographic data for 20-epiphyllostacin N (9): M = 378.45, monoclinic, a = 14.7955(3) Å, b = 6.6095(2) Å, c = 19.9799(5) Å, α = 90.00°, β = 90.0360(10)°, γ = 90.00°, V = 1953.85(9) Å3, T = 100(2) K, space group P2, Z = 4, μ(Cu Kα) = 0.764 mm−1, 9056 reflections measured, 4455 independent reflections (Rint = 0.0426). The final R1 values were 0.2256 (I > 2σ(I)). The final wR(F2) values were 0.5009 (I > 2σ(I)). The final R1 values were 0.2314 (all data). The final wR(F2) values were 0.5113 (all data). The goodness of fit on F2 was 2.628. Flack parameter = 0.9(7).

Cytotoxicity Assays. Five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480; obtained from ATCC, Manassas, VA, USA) were used in the cytotoxicity assay. All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), which were supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere containing 5% CO2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of MTS (Sigma, St. Louis, MO, USA).36 Briefly, cells were seeded into each well of a 96-well cell culture plate. After 12 h of incubation at 37 °C, the test compound (40 μM) was added. After incubation for 48 h, the cells were subjected to the MTS assay. Compounds with a growth inhibition rate of 50% were further evaluated at various concentrations in triplicate for 48 h; cis-platin was used as the positive control. The IC50 value of each compound was calculated according to the Reed and Muench method.37 Nitric Oxide Production in RAW264.7 Macrophages. RAW264.7 cells were seeded in 96-well cell culture plates (2 × 105 cells/well) and treated with serial dilutions of the compounds at a maximum concentration of 25 μM; this treatment was followed by stimulation with LPS (1 μg/mL) for 18 h. NO production in the supernatant was assessed using Griess reagents. The absorbance at 550 nm was measured using a 2104 Envision multilabel plate reader (PerkinElmer Life Sciences, Inc., Boston, MA, USA). MG-132 was used as a positive control.38 The viability of RAW264.7 cells was simultaneously evaluated using the MTT assay to exclude the interference of the cytotoxicity of the test compounds. The absorbance was measured at 595 nm.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00802. 1 H and 13C NMR, DEPT, HSQC, HMBC, COSY, NOESY, HREIMS, IR, and UV spectra of compounds 1, 8, and 9; 1H and 13C NMR, DEPT, and HREIMS spectra of compounds 2−7 and 10−13; X-ray data of compounds 1, 8, and 9 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ⊥

J. Yang and W.-G. Wang contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported financially by the National Natural Science Foundation of China (Grants 21322204 and 81172939), the NSFC-Joint Foundation of Yunnan Province (Grant U1302223), the reservation-talent project of Yunnan Province (Grant 2011CI043), and the West Light Foundation of the Chinese Academy of Sciences (J.-X.P.).

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REFERENCES

(1) Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita. Flora Reipublicae Popularis Sinicae; Science Press: Beijing, 1977; Vol. 66, pp 416−418. (2) (a) Sun, H.-D.; Huang, S.-X.; Jiang, B. Diterpenoids from Isodon Species; Science Press: Beijing, 2001. (b) Sun, H.-D.; Huang, S.-X.; Han, Q.-B. Nat. Prod. Rep. 2006, 23, 673−698.

H



Article

(25) Yoshio, T.; Takashi, M.; Hideaki, O. J. Nat. Prod. 1994, 57, 650−653. (26) Sun, H.-D.; Lin, Z.-W.; Xu, Y.-L.; Minami, Y.; Marunaka, T.; Togo, T.; Takeda, Y.; Fujita, T. Heterocycles 1986, 24, 1−4. (27) Han, Q.-B.; Li, S.-H.; Peng, L.-Y.; Sun, H.-D. Heterocycles 2003, 60, 933−938. (28) Li, L.-M.; Li, G.-Y.; Pu, J.-X.; Xiao, W.-L.; Ding, L.-S.; Sun, H.-D. J. Nat. Prod. 2009, 72, 1851−1856. (29) (a) Ke, Y.-K.; Dong, H.-R. Handbook of Analytical Chemistry (Second ed.); Chemical Industry Press: Beijing, 1998; Third Book, pp 944; 949; 955; 958. (b) Wang, W.-G.; Li, X.-N.; Du, X.; Wu, H.-Y.; Liu, X.; Su, J.; Li, Y.; Pu, J.-X.; Sun, H.-D. J. Nat. Prod. 2012, 75, 1102− 1107. (30) Flack, H. D. Acta Crystallogr., Sect. A: Found. Crystallogr. 1983, 39, 876−881. (31) Hooft, R. W. W.; Straver, L.-H.; Spek, A.-L. J. Appl. Crystallogr. 2008, 41, 96−103. (32) Li, B.-L.; Tian, X.-H. Phytochemistry 2001, 58, 543−546. (33) Node, M.; Sai, M.; Fuji, K.; Fujita, E.; Takeda, S.; Unemi, N. Chem. Pharm. Bull. 1983, 31, 1433−1436. (34) McCartney-Francis, N.-L.; Song, X.; Mizel, D.-E.; Wahl, S.-M. J. Immunol. 2001, 166, 2734−2740. (35) Sheldrick, G.-M.; Schneider, T.-R. Methods Enzymol. 1997, 277, 319−343. (36) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A. J. Natl. Cancer Inst. 1991, 83, 757−766. (37) Reed, L.-J.; Muench, H. Am. J. Hyg. 1938, 27, 493−497. (38) Fan, J.-T.; Su, J.; Peng, Y.-M.; Li, Y.; Li, J.; Zhou, Y.-B.; Zeng, G.-Z.; Yan, H.; Tan, N.-H. Bioorg. Med. Chem. 2010, 18, 8226−8234.

(3) (a) Yang, J.-H.; Wang, W.-G.; Du, X.; He, F.; Zhang, H.-B.; Li, X.N.; Li, Y.; Pu, J.-X.; Sun, H.-D. J. Nat. Prod. 2014, 77, 2444−2453. (b) Wu, H.-Y.; Zhan, R.; Wang, W.-G.; Jiang, H.-Y.; Du, X.; Li, X.-N.; Li, Y.; Pu, J.-X.; Sun, H.-D. J. Nat. Prod. 2014, 77, 931−941. (c) Jiang, H.-Y.; Wang, W.-G.; Zhou, M.; Wu, H.-Y.; Zhan, R.; Li, X.-N.; Du, X.; Li, Y.; Pu, J.-X.; Sun, H.-D. J. Nat. Prod. 2013, 76, 2113−2119. (d) Yang, J.-H.; Du, X.; He, F.; Zhang, H.-B.; Li, X.-N.; Su, J.; Li, Y.; Pu, J.-X.; Sun, H.-D. J. Nat. Prod. 2013, 76, 256−264. (e) Wang, W.G.; Yan, B.-C.; Du, X.; Li, X.-N.; Wu, H.-Y.; Zhan, R.; Li, Y.; Pu, J.-X.; Sun, H.-D. Tetrahedron 2014, 70, 7445−7453. (f) Zhan, R.; Li, X.-N.; Du, X.; Wang, W.-G.; Dong, K.; Su, J.; Li, Y.; Pu, J.-X.; Sun, H.-D. J. Nat. Prod. 2013, 76, 1267−1277. (4) Yang, J.; Jiang, H.; Wang, C.-Y.; Yang, B.; Zhao, L.-J.; Hu, D.-L.; Qiu, G.-H.; Dong, X.-L.; Xiao, B. Biomed. Pharmacother. 2015, 72, 125−134. (5) Li, L.; Yue, G.-G.-L.; Pu, J.-X.; Sun, H.-D.; Fung, K.-P.; Leung, P.C.; Han, Q.-B.; Lau, C.-B.-S.; Leung, P.-S. Curr. Mol. Med. 2014, 14, 673−689. (6) (a) Yao, R.; Chen, Z.-L.; Zhou, C.-C.; Luo, M.; Shi, X.-J.; Li, J.-G.; Gao, Y.-B.; Zhou, F.; Pu, J.-X.; Sun, H.-D.; He, J. J. Nat. Prod. 2015, 78, 10−16. (b) Aquila, S.; Weng, Z.-Y.; Zeng, Y.-Q.; Sun, H.-D.; Rios, J.-L. J. Nat. Prod. 2009, 72, 1269−1272. (7) (a) Li, D.-H.; Xu, S.-T.; Cai, H.; Pei, L.-L.; Zhang, H.-Y.; Wang, L.; Yao, H.-Q.; Wu, X.-M.; Jiang, J.-Y.; Sun, J.-Y.; Xu, J.-Y. Eur. J. Med. Chem. 2013, 64, 215−221. (b) Wang, L.; Li, D.-H.; Xu, S.-T.; Cai, H.; Yao, H.-Q.; Zhang, H.-Y.; Jiang, J.-Y.; Xu, J.-Y. Eur. J. Med. Chem. 2012, 52, 242−250. (c) Wang, Z.; Xu, Z.-J.; Niu, Z.-C.; Liang, B.-J.; Niu, J. Cell Biochem. Biophys. 2015,73, 55910.1007/s12013-015-0687-4. (8) Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita. Flora Reipublicae Popularis Sinicae; Science Press: Beijing, 1977; Vol. 66, p 440. (9) Li, X.; Xiao, W.-L.; Huang, S.-X.; Weng, Z.-Y.; Shen, Y.-H.; Han, Q.-B.; Sun, H.-D. Helv. Chim. Acta 2006, 89, 1181−1186. (10) Fujita, T.; Sun, H.-D.; Yoshio, T.; Yoshinori, M.; Teruyoshi, M.; Lin, Z.-W.; Xu, Y.-L. J. Chem. Soc., Chem. Commun. 1985, 1738−1739. (11) Chen, Y.-P.; Sun, L.-P.; Sun, H.-D. Acta Bot. Yunnan. 1991, 3, 331−334. (12) Hou, A.-J.; Yang, H.; Jiang, B.; Zhao, Q.-S.; Lin, Z.-W.; Sun, H.D. Fitoterapia 2000, 71, 417−419. (13) Li, X.; Pu, J.-X.; Li, S.-H.; Huang, S.-X.; Weng, Z.-Y.; Han, Q.-B.; Sun, H.-D. Heterocycles 2007, 71, 2441−2447. (14) Li, X.; Weng, Z.-Y.; Li, Y.; Pu, J.-X.; Huang, S.-X.; Xiao, W.-L.; Sun, H.-D. Helv. Chim. Acta 2008, 91, 1130−1136. (15) Li, X.; Xiao, W.-L.; Pu, J.-X.; Ban, L.-L.; Shen, Y.-H.; Weng, Z.Y.; Li, S.-H.; Sun, H.-D. Phytochemistry 2006, 67, 1336−1340. (16) Yan, F.-L.; Zhang, L.-B.; Zhang, J.-X.; Sun, H.-D. Chin. Chem. Lett. 2007, 18, 1383−1385. (17) Zhao, A.-H.; Zhang, Y.; Xu, Z.-H.; Liu, J.-W.; Jia, W. Helv. Chim. Acta 2004, 87, 3160−3166. (18) Li, Y.; Hua, S.-M.; Xue, D.-Y.; Chen, Y.-Z. Chem. J. Chin. Univ. 1990, 11, 1222−1226. (19) (a) Chen, Y.-Z.; Bai, G.; Meng, X.-J. Acta Chim. Sin. 1989, 6, 549−553. (b) Fujita, E.; Fujita, T.; Shibuya, M. Tetrahedron Lett. 1966, 27, 3153−3162. (c) Fujita, E.; Fujita, T.; Shibuya, M. Chem. Pharm. Bull. 1968, 16, 509−515. (20) Huang, H.; Zhang, H.-J.; Sun, H.-D. Phytochemistry 1990, 29, 2591−2595. (21) Chao, J.-H.; Zhao, H.-Q.; Wang, H.-Q.; Sun, H.-D. Acta Bot. Yunnan. 1983, 5, 311−314. (22) Sun, H.-D.; Qiu, S.-X.; Lobkovsky, E.-B.; Lin, L.-Z.; Farnsworth, N.-R.; Clardy, J.; Fong, H. Tetrahedron 2001, 57, 65−70. (23) (a) Fujita, E.; Fujita, T.; Shibuya, M. Tetrahedron Lett. 1966, 27, 3153−3162. (b) Fujita, E.; Fujita, T.; Shibuya, M. Chem. Commun. 1966, 10, 297. (c) Fujita, E.; Fujita, T.; Fuji, K.; Ito, N. Tetrahedron 1966, 22, 3423−3441. (24) (a) Kanatomo, S.; Shinichiro, S. Yakugaku Zasshi 1961, 81, 1807−1808. (b) Takeda, Y.; Takeda, K.; Fujita, T. J. Chem. Soc., Perkin Trans. 1 1986, No. 4, 689−694. I


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