Dimeric Abietane Diterpenoids and Sesquiterpenoid Lactones from

Article pubs.acs.org/jnp

Dimeric Abietane Diterpenoids and Sesquiterpenoid Lactones from Teucrium viscidum Chun Gao,† Li Han,‡ Dan Zheng,‡ Hongwei Jin,† Chunyan Gai,† Jianbin Wang,† Hao Zhang,† Liangren Zhang,† and Hongzheng Fu*,† †

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People’s Republic of China ‡ Institute of Microbial Pharmaceuticals, College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China S Supporting Information *

ABSTRACT: A new abietane diterpenoid, teuvisone (2), a pair of new dimeric abietane diterpenoid stereoisomers, biteuvisones A (3) and B (4), and three new sesquiterpenoid lactones, teuvislactones A−C (6, 7, and 10), were isolated from the whole plants of Teucrium viscidum, along with four known terpenoids (1, 5, 8, and 9). The structures of the new compounds were elucidated by spectroscopic analysis, and the absolute configurations of 5−10 were determined by electronic circular dichroism analysis. The isolated compounds were evaluated for their cytotoxic effects against five human cancer cell lines and for their α-glucosidase inhibitory effects.

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Teucrium viscidum Blume (Lamiaceae) is a perennial herb, widely distributed throughout northwest mainland China, Japan, North Korea, Myanmar, India, Indonesia, and the Philippines. The whole parts of this plant have been used for centuries in traditional Chinese medicine for the treatment of rheumatoid arthritis, hematemesis, pulmonary abscesses, traumatic injuries, acute gastroenteritis, and venomous snakebites.1 Only a few studies have been conducted on the chemical constituents of T. viscidum, and efforts to date on this plant have culminated in the isolation of clerodane diterpenoids,2 glucosylated coumaroyltyramine derivatives,3 ursane-type triterpenoids, lignans, steroids,4 guaiane sesquiterpenoids, and several compounds of miscellaneous types.5 In an ongoing search for new bioactive terpenoids from Chinese medicinal plants, a new abietane diterpenoid, teuvisone (2), a pair of new dimeric abietane diterpenoid stereoisomers, biteuvisones A (3) and B (4), three new sesquiterpenoid lactones, teuvislactones A−C (6, 7, and 10), a known abietane diterpenoid (1), and three known sesquiterpenoid lactones (5, 8, and 9) were obtained from the whole plants of T. viscidum. Herein, the isolation and structural elucidation of these compounds, as well as an evaluation of their caner cell line cytotoxic activities and α-glucosidase inhibitory effects, are reported. The spectroscopic data of 1 are documented for the first time, and the absolute configurations of known compounds 5, 8, and 9 are defined. A plausible biosynthetic pathway for the genesis of compounds 3 and 4 is proposed. © XXXX American Chemical Society and American Society of Pharmacognosy

RESULTS AND DISCUSSION

The NMR data analysis of 1 suggested that it is the known compound 2,11,12-trihydroxy-7,20-epoxy-8,11,13-abietatriene. 6 No detailed assignments of the NMR data or the relative configuration determination for this compound have been reported previously. Its NMR assignments (Table 1) are documented for the first time, and its relative configuration was confirmed by single-crystal X-ray diffraction analysis (Figure 1). Teuvisone (2) was isolated as a yellow, amorphous solid. The molecular formula was determined as C20H26O4 from the 13 C NMR and HRESIMS data, showing a molecular ion at m/z 353.1722 [M + Na]+. The IR spectrum of 2 showed absorption bands characteristic of hydroxy (3411 cm−1) and carbonyl (1712 cm−1) groups. The 1H and 13C NMR data (Table 1) revealed structural features similar to those of przewalskin G,7 except for an extra hydroxy group (δH 4.52) at C-2 (δC 62.5) in 2. Compound 2 was assigned the same relative configuration as 1 by analysis of its NOESY spectrum. The correlations of H-5 (δH 1.27) with H-1α (δH 2.19) and H3-18α (δH 0.82) suggested that they occupy the same face of the molecule. The crosspeaks of H-2 (δH 3.64) with H3-19β (δH 1.02) and H-20a (δH 4.05) indicated their spatial proximity. Thus, the structure of compound 2 was assigned as 2α-hydroxy-7β,20-epoxy-11,12dione-8,13-abietadiene. Received: September 24, 2014

A

DOI: 10.1021/np500746n J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H and 13C NMR Spectroscopic Data (400 MHz, DMSO-d6) for Compounds 1 and 2 1 position

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

1α

40.0, CH2

2.54, dd (12.8, 12.2) 2.15, br d (12.8)

38.2, CH2

2

63.1, CH

62.5, CH

3α 3β

50.4, CH2

3.71, ddd (12.2, 11.8, 3.4) 1.10, ma 1.70, dt (11.8, 2.8)

2.19, dd (13.5, 12.7) 2.00, br d (12.7) 3.64, m

4 5

34.5, C 42.7, CH

6α

29.5, CH2

1β

6β 7 8 9 10 11 12 13 14 15 16 17 18α 19β 20a 20b OH-2 OH-11 OH-12

Biteuvisone A (3) was obtained as a yellow, amorphous powder, and its molecular formula of C40H52O8 was deduced from the 13C NMR and HRESIMS data. The IR spectrum of 3 showed absorption bands characteristic of hydroxy and carbonyl groups at 3335 and 1738 cm−1, respectively. The NMR data of 3 (Table 2) revealed the presence of two abietane-type diterpenoid moieties, indicating it to be a dimeric abietane diterpenoid. One of the moieties (fragment A, Figure 3) produced signals characteristic of a conjugated α-diketo group (δH 7.08, δC 197.6, 185.0, 145.3, 149.1), two oxygenated methine groups (δH 4.55, 3.96, δC 71.8, 63.2), an oxygenated methylene (δH 4.33, 4.17, δC 65.2), and an isopropyl group (δH 2.78, 0.98, 1.06, δC 27.5, 20.1, 21.8). These data suggested that fragment A possesses a structure similar to teuvisone (2), except that the sp2 carbons (C-8, C-9 at δC 150.9 and δC 139.0) in 2 were replaced by two sp3 oxygenated tertiary carbons (δC 84.9 and 87.9) in fragment A. The structure of this fragment was confirmed by the HMBC correlations from H-7 (δH 4.55) to C-8 (δC 84.9) and C-9 (δC 87.9) and from H-14 (δH 7.08) to C-9 (δC 87.9). The 1H and 13C NMR data of fragment B (Figure 3) were similar to those of 1, except that the signals of C-8 and C-13 were deshielded by Δ 4.1 and 3.3 ppm, respectively. The detailed NMR data analysis (Figure 2) indicated that fragment B has the same structure as 1 except for the two phenolic hydrogens. Therefore, fragments A and B of 3 contributed 40 carbon atoms and 10 oxygen atoms, with 14 indices of hydrogen deficiency. Considering the overall formula of 3 (C40H52O8), the two redundant oxygen atoms could be the result of two shared oxygen atoms, and the missing one index of hydrogen deficiency could be attributed to the cyclic linkage between these two subunits. All of these data permitted the connection of the two moieties at C-8/C-11′, C-9/C-12′ or C-8/C-12′, C9/C-11′ through an oxygen bridge. No HMBC correlations were found to determine the linkage of the two fragments

2

a

69.8, CH 132.3, 128.2, 40.7, 140.7, 142.0, 133.1, 111.2, 26.1, 22.9, 22.9, 32.9, 22.0, 68.6,

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

1.24, dd (11.5, 5.3) 1.84, ddd (13.2, 5.3, 3.9) 1.39, dd (13.2, 11.5) 4.60, dd (3.9, 1.5)

6.52, 3.22, 1.10, 1.12, 0.81, 1.06, 4.06, 2.89, 4.48, 7.72, 8.04,

s sept (6.8) d (6.8) d (6.8) s s d (8.5) d (8.5) d (3.4) s s

49.8, CH2

34.2, C 40.9, CH 27.0, CH2

68.7, CH 150.9, 139.0, 41.2, 176.6, 179.6, 147.9, 132.4, 26.9, 21.2, 21.3, 32.9, 21.6, 67.2,

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

1.03, ma 1.67, m

1.27, dd (12.0, 6.4) 1.82, ddd (14.0, 6.4, 4.0) 1.56, dd (14.0, 12.0) 4.58, br s

6.90, 2.78, 1.03, 1.04, 0.82, 1.02, 4.05, 2.97, 4.52,

s sept (6.8) d (6.8) d (6.8) s s d (8.5) d (8.5) br s

Overlapping signals.

because there are no hydrogen atoms at C-8, C-9, C-11′, and C-12′ in 3. Thus, four possible structures (P1−4) were proposed by the rational linkage of fragments A and B (Figure 3). A simulation of the stereostructures of P1−4 (Figure 4) revealed that H-1 and H-1′ in P1 and P2 are sufficiently close to produce an NOE effect. In contrast, the spatial distances between H-1 and H-1′ in P3 and P4 are greater than 5 Å. The spatial distance between H-6α and H-14 is 3.1 Å in P1, which is approximately 1.0 Å less than that of P2 (Figure 4), thus affording a stronger NOE correlation between H-6α and H-14 in P1 than in P2. The NOESY experiment of 3 showed diagnostic correlations between H-1 (δH 2.59, 1.78) and H-1′α (δH 3.57) and between H-6α (δH 1.89) and H-14 (δH 7.08), suggesting that P1 is the most likely structure for 3. To confirm the structure of 3, the key issue was the definition of the configuration of the C-8 and C-9 stereogenic centers. The electronic circular dichroism (ECD) spectra of (2S,5S,7S,8S,9R,10S,2′S,5′S,7′S,10′R)-P1 and its 8,9-diastereoisomer (2S,5S,7S,8R,9S,10S,2′S,5′S,7′S,10′R)-P2 were calculated using the TDDFT method at the B3 LYP/6-31+G(d) level. The calculated ECD spectrum of P1 showed the same pattern as the experimental ECD spectrum of 3 and was B



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determined by a comparison of the experimental and calculated ECD spectra of (1R,3S,4R,5R,8R,10S)-5 and its enantiomer. The experimental ECD spectrum of 5 fits well with the calculated ECD spectrum of (1R,3S,4R,5R,8R,10S)-5 and was opposite that of its enantiomer (Figure 6). Therefore, the absolute configuration of 5 was established as shown. The molecular formula of teuvislactone A (6) was determined as C17H24O6 from the 13C NMR data and the [M − H]− ion at m/z 323.1503 in the HRESIMS. The 1H and 13C NMR data (Table 3) of this compound were similar to those of 5, except for a C-1 acetyl group in 6 instead of the C-3 acetyl group in 5. The presence and position of this group were confirmed by the HMBC correlations from H-1 (δH 4.95) and H3-17 (δH 1.89) to the acetyl carbonyl carbon (δC 170.5). The absolute configuration of 6 was assigned as identical to that of 5 based on the similarities between their ECD spectra (Figure 7). Thus, the structure of 6 was defined as (1R,3S,4R,5R,8R,10S)1-acetoxy-3,8-dihydroxyeudesm-7(11)-en-8,12-olide. Teuvislactone B (7) was assigned a molecular formula of C15H22O5 according to the 13C NMR data and by HRESIMS analysis (m/z 565.3000, [2 M + H]+). A comparison of the NMR data of 7 (Table 3) with those of 5 suggested that 7 was the deacetyl derivative of 5. This assertion was confirmed by the presence of a hydrogen signal at δH 4.81 (OH-3) and the HMBC correlations from OH-3 (δH 4.81) to C-2 (δC 33.8), C3 (δC 71.7), and C-4 (δC 35.9). The absolute configuration of 7 was assigned as being identical to that of 5 based on the similarities between their ECD spectra (Figure 7). Therefore, the structure of 7 was determined as (1R,3S,4R,5R,8R,10S)1,3,8-trihydroxyeudesm-7(11)-en-8,12-olide. Compound 8 was found to possess the same planar structure and relative configuration as 1α-acetoxy-8α-hydroxy-2-oxoeudesman-3,7(11)-dien-8,12-olide (8a).9 The absolute configuration of 8 was defined by comparison of its experimental ECD spectrum (Figure 7) with that of 8a. The experimental ECD spectrum of 8 exhibited negative Cotton effects at 320 (Δε −0.7), 262 (Δε −1.2), and 210 (Δε −16.2) nm and a positive Cotton effect at 238 (Δε +4.5) nm, which were opposite those of 8a [ECD (MeCN, Δε) λmax 327 (+0.7), 257 (+l.l), 234 (−6.7), 212 (+10.9) nm],9 suggesting that compound 8 is the enantiomer of 8a. Therefore, its absolute configuration was defined as 1S, 5S, 8S, and 10R. The structure of 8 was determined as (−)-1S,5S,8S,10R-1-acetoxy-8-hydroxy2-oxoeudesman-3,7(11)-dien-8,12-olide. The NMR data of 9 were consistent with those of 1α,8αdihydroxy-2-oxoeudesman-3,7(11)-dien-8,12-olide (9a), 9 which revealed that 9 and 9a possess the same relative configuration. There has been no report on the specific rotation value of 9a. The ECD spectrum of 9 (Figure 7) was similar to that of 8 and opposite that of 8a, indicating that the absolute configuration of 9 is the same as that of 8. Therefore, the structure of 9 was determined as (−)-1S,5S,8S,10R-1,8dihydroxy-2-oxoeudesman-3,7(11)-dien-8,12-olide. Teuvislactone C (10) gave a molecular formula of C17H20O7 as determined by the 13C NMR data and from an HRESIMS ion at m/z 335.1127 [M − H]−. A comparison of the 1H and 13 C NMR data of 10 (Table 3) with those of 8 suggested that an extra hydroxy group (δH 7.68) is present at C-5 (δC 77.4) in 10. The absolute configuration of this compound was assigned as being identical to that of 8, based on the similarities between their ECD spectra (Figure 7). Thus, the structure of 10 was determined as (−)-1S,5R,8S,10S-1-acetoxy-5,8-dihydroxy-2-oxoeudesman-3,7(11)-dien-8,12-olide.

Figure 1. ORTEP diagrams of compounds 1 and 5.

generally opposite that of P2 (Figure 5). Thus, the structure of 3 was established as shown. Biteuvisone B (4) was isolated as a yellow, amorphous powder and had the same molecular formula, C40H52O8, as 3. The close resemblance between the NMR data of 4 and 3 indicated that 4 is an isomer of 3. The only difference between these compounds was the pattern of the connection of fragments A and B. The diagnostic NOESY correlation between H-1α (δH 2.46) and H-1′α (δH 3.99) of 4 suggested that their spatial proximity is identical to those in P1 and P2. In addition, the absence of a cross-peak between H-6α (δH 2.37) and H-14 (δH 7.21) suggested that P2 is the rational structure for 4. Using this result and the similarity between the calculated ECD spectrum of P2 and the experimental ECD spectrum of 4 (Figure 5), the structure of 4 was determined as shown for P2. A literature review has revealed that approximately 20 dimeric abietane diterpenoids have been reported since 2000.8 Hetero-Diels−Alder reactions are presumed to be key steps in the biosynthetic pathway for several of these compounds. Thus, a plausible biosynthetic pathway is proposed in Figure 3. Compounds 3 and 4 are formed through a hetero-Diels−Alder reaction of the o-quinone of teuvisone. Considering the steric effects in the transition state, the less sterically hindered products 3 (P1) and 4 (P2) would be two major products. The result of the biosynthetic analysis was supported by the fact that P3 and P4 were not found experimentally. Compound 5, 3-acetoxy-1,8-dihydroxyeudesm-7(11)-8,12olide, has been reported by Xiong et al.,6 but its absolute configuration has not been defined. In this study, the relative configuration of 5 was confirmed through X-ray diffraction analysis (Figure 1), and its absolute configuration was C



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Table 2. 1H and 13C NMR Spectroscopic Data (400 MHz, Pyridine-d5) for Compounds 3 and 4 3

4

position

δC, type

δH (J in Hz)

position

δC, type

1α

37.6 CH2

2.59, br d (13.6) 1.78, dd (13.6,11.8) 3.96, tt (11.8, 3.6) 1.21, ma 1.98, ma

1′α

41.0 CH2

1β 2

63.2 CH

3α 3β 4 5 6α 6β 7 8 9 10 11 12 13 14 15

50.7 CH2 35.4 C 38.7 CH 25.5 CH2 71.8 84.9 87.9 44.6 197.6 185.0 149.1 145.3 27.5

CH C C C C C C CH CH

position

δC, type

δH (J in Hz)

position

δC, type

δH (J in Hz)

1α

37.3 CH2

2.46, dd (13.8,11.7) 1.82, dd (13.8, 4.1) 4.11, tt (11.7, 4.1) 1.50, ma 2.02, ma

1′α

40.0 CH2

3.99, dd (13.9,12.0) 2.68, ma

2′

64.4 CH

3′α 3′β 4′ 5′ 6′α 6′β 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′

50.7 CH2

4.35, tt (12.0, 3.8) 2.00, ma 2.17, ma

3.57, br d (12.8) 2.77, ma

1β

2′

64.2 CH

4.33, ma

2

63.6 CH

51.2 CH2

1.50, ma 2.15, ma

3α 3β 4 5 6α 6β 7 8 9 10 11 12 13 14 15

50.0 CH2

7.08, s 2.78, ma

3′α 3′β 4′ 5′ 6′α 6′β 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′

1.53, 1.89, 2.10, 4.55,

ma ma ma br s

35.1 C 42.9 CH 30.0 CH2 71.0 136.4 131.9 41.9 142.4 142.2 136.4 115.3 26.6


16

20.1 CH3

0.98, d (6.9)

16′

23.4 CH3

17

21.8 CH3

1.06, d (6.9)

17′

23.1 CH3

18α 19β 20a

32.6 CH3 23.0 CH3 65.2 CH2

0.74, s 1.09, s 4.33, d (9.6)

18′α 19′β 20′a

33.2 CH3 22.9 CH3 68.4 CH2

4.17, d (9.6)

20′b

20b a

1′β

δH (J in Hz)

1.17, 2.04, 1.44, 4.91,

ma ma ma br s

6.94, s 3.44, sept (6.9) 1.25, d (6.9) 1.19, d (6.9) 0.84, s 1.28, s 4.60, d (8.5) 3.73, d (8.5)

35.2 C 36.4 CH 22.7 CH2 72.3 82.9 87.7 43.6 197.8 185.2 148.5 150.2 27.6

CH C C C C C C C CH

2.54, 2.37, 1.97, 4.42,

ma ma ma br s

16

20.4 CH3

7.21, s 2.66, sept (6.9) 0.83, d (6.9)

17

21.7 CH3

18α 19β 20a

32.9 CH3 21.7 CH3 66.4 CH2

20b

1′β

35.0 C 42.2 CH 29.6 CH2 70.7 136.3 132.2 42.1 142.5 141.8 136.5 116.0 26.7


1.88, 1.98, 1.23, 4.89,

ma ma ma br s

16′

23.2 CH3

7.01, s 3.45, sept (6.9) 1.28, d (6.9)

1.04, d (6.9)

17′

24.2 CH3

1.27, d (6.9)

0.85, s 1.12, s 4.25, d (11.8)

18′α 19′β 20′a

33.2 CH3 22.4 CH3 69.2 CH2

0.87, s 1.22, s 4.43, d (8.8)

3.51, dd (11.8, 1.3)

20′b

3.20, d (8.8)

Overlapping signals.

commonly associated with bicyclic sesquiterpenoid lactones.11 Only compounds 9 and 10 showed weak α-glucosidase inhibitory effects, with IC50 values of 568.3 and 299.8 μM, respectively. Acarbose was used as a positive control and had an IC50 value of 169.3 μM. Compounds 1−8 were inactive (IC50 > 1000 μM) in the same assay. The results of the current study suggest that the α,β-unsaturated carbonyl and free hydroxy moieties of ring A may be essential for the α-glucosidase inhibitory effects of the sesquiterpenoid lactones.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an X-5 melting point apparatus (Gongyi City Yuhua Instrument Co., Ltd., Gongyi, People’s Republic of China). Optical rotations were obtained using an Autopol III polarimeter. Ultraviolet (UV) data were obtained using a Varian Cary 300 ultraviolet−visible spectrophotometer (Palo Alto, CA, USA). Electronic circular dichroism spectra were recorded in MeOH on a JASCO J-810 spectropolarimeter (JASCO, Hachioji, Tokyo, Japan). Infrared (IR) data were recorded as KBr disks on a Nicolet 470 FT-IR spectrophotometer (Nicolet, Madison, WI, USA). 1 H (400 MHz),13C (100 MHz), and 2D NMR spectra were obtained with TMS as an internal standard on a Bruker AV 400 spectrometer (Bruker, Karlsruhe, Baden−Wuerttemberg, Germany). HRESIMS data were obtained using a Bruker Daltonics APEX IV Fourier transform ICR high-resolution mass spectrometer (Bruker). Crystal data were obtained on a Rigaku MicroMax 002+ single-crystal X-ray diffractometer with the wavelength for Cu Kα irradiation of 1.5418 Å. Open

Figure 2. (a) COSY (bold lines) and selected HMBC (blue arrows) correlations of 3 and 4 and (b) selected NOESY (black arrows) correlations of 3.

Compounds 1−10 were tested for their cytotoxic effects because abietane diterpenoids have been reported to be cytotoxic against a broad range of different cell lines.10 Five human cancer cell lines were used, namely, H460, HepG2, BGC823, HCT116, and HeLa cells (Table 4). Compound 4 exhibited IC50 values over the range 4.8−8.5 μM against the five different cell lines. Compounds 1 and 3 and the sesquiterpenoid lactones 5−10 were inactive against the five cell lines used, with IC50 values of >10 μM. Compounds 1−10 were also tested for their α-glucosidase inhibitory effects, which are D



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Figure 3. Plausible biogenetic pathway for the genesis of 3 and 4. dissolved in MeOH, and the resulting solution was extracted with petroleum ether. The MeOH fraction (156.0 g) was subjected to CC over silica gel eluting with a gradient of CH3Cl and MeOH (100:1 to 0:1 v/v) to yield 10 fractions (F1−10). Fraction F4 (6.2 g) was separated by chromatography over a Sephadex LH-20 column eluting with MeOH to afford eight fractions (F4.1−4.8). Fraction F4.3 (574.5 mg) was separated over an ODS column eluting with a gradient of MeOH and H2O (40−90%) and further purified by preparative HPLC (MeOH−H2O, 68%) to afford compounds 1 (68.4 mg), 2 (13.3 mg), 3 (10.8 mg), and 4 (9.3 mg). Fraction F3 (7.3 g) was purified by CC over silica gel eluting with a gradient of petroleum ether and EtOAc (5:1 to 1:2) to afford nine fractions (F3.1−3.9). F3.7 (884.2 mg) was purified over a Sephadex LH-20 column eluting with MeOH to afford six subfractions (F3.7.1− 3.7.6). Fraction F3.7.5 (233.7 mg) was purified over an ODS column eluting with a gradient of MeOH and H2O (30−90%) before further purification by preparative HPLC (MeOH−H2O, 40%) to afford compounds 5 (87.6 mg) and 9 (6.1 mg). Fraction F2 (4.3 g) was purified by CC over silica gel eluting with a gradient of petroleum ether and EtOAc (8:1 to 1:1) to afford 11 fractions (F2.1−2.11). F2.10 (560.3 g) was separated on Sephadex LH-20 CC (MeOH) to afford four subfractions (F2.10.1−2.10.4). Compound 6 (6.3 mg) was obtained from fraction F2.10.2 (56.1 mg) by ODS (MeOH−H2O, 30−80%) and further purified by preparative

column chromatography (CC) separations were performed over silica gel (200−300 mesh; Qingdao Marine Chemical Co., Qingdao, People’s Republic of China), ODS (50 μm, YMC, Japan), and Sephadex LH-20 (Taizhou Luqiao Sijia Chemical Reagents Factory, Taizhou, People’s Republic of China). Reversed-phase preparative high-performance liquid chromatography (HPLC) was performed on a LabAlliance system equipped with a LabAlliance series III pump, model 2000 ultraviolet−visible detector, and a YMC-pack C18 column (10 × 250 mm). TLC was conducted with glass precoated with silica gel GF254 (Qingdao Marine Chemical Co.). The MTT assay and Griess reaction were analyzed using a microplate reader (BioTek Synergy H1, BioTek Instruments, Inc., Winooski, VT, USA). Plant Material. The dried whole plants of T. viscidum were purchased in March 2012 from the Chinese Herbal Medicine Market in Qingyuan City, Guangzhou Province, People’s Republic of China, and were identified by Prof. Shixia Guan of the School of Guangzhou University of Chinese Medicine, Guangzhou, People’s Republic of China. A voucher specimen (No. 201210XJC) was deposited in the State Key Laboratory of Natural and Biomimetic Drugs (Peking University). Extraction and Isolation. The dried whole plants of T. viscidum (10 kg) were extracted with 95% EtOH (3 × 30 L) under reflux (4 h each). The combined EtOH extracts were concentrated under vacuum to afford a crude extract (1555 g), which was suspended in hot H2O and extracted with EtOAc. The EtOAc fraction (181.4 g) was E



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Figure 4. Low-energy conformers of P1−4 and the key NOESY correlations of P1−2.

Figure 5. Comparison between calculated and experimental ECD spectra of 3 and 4.

Figure 6. Comparison between the calculated and experimental ECD spectra of 5. Fraction F5 (3.8 g) was purified using Sephadex LH-20 CC with MeOH to yield eight fractions (F5.1−5.8). Compound 7 (15.4 mg)

HPLC (MeOH−H2O, 36%). Compound 8 (26.7 mg) was obtained from fraction F2.10.3 (120.5 mg) by ODS (MeOH−H2O, 30−80%). F



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Table 3. 1H and 13C NMR Spectroscopic Data for Compounds 6 and 10 (400 MHz, Pyridine-d5) and Compound 7 (400 MHz, DMSO-d6) 6

a

position

δC, type

1 2α 2β 3 4 5 6α 6β 7 8 9α 9β 10 11 12 13 14 15 16 17 OH-1 OH-3 OH-5 OH-8

76.8 CH 32.8 CH2 70.0 36.3 40.6 24.6

CH CH CH CH2

161.7 C 105.1 C 46.5 CH2 39.1 121.2 172.6 8.0 17.7 16.6 170.5 21.2

C C C CH3 CH3 CH3 C CH3

7 δH (J in Hz) 4.95, 2.14, 2.38, 3.96, 1.80, 2.39, 2.34, 2.84,

br s dt (15.6, 3.4) ma s m ma ma dd (12.3, 2.3)

2.37, ma 2.36, ma

1.68, s 1.47, s 1.26, d (6.7)

δC, type 74.9 CH 33.8 CH2 71.7 35.9 40.3 24.2

CH CH CH CH2

161.8 C 105.1 C 46.0 CH2 39.9 120.3 172.2 8.3 17.2 16.6

C C C CH3 CH3 CH3

10 δH (J in Hz) 3.28, 1.75, 1.96, 3.66, 1.64, 1.56, 1.98, 2.66,

m ma ma m m ma ma dd (13.0, 3.1)

1.94, ma 1.77, ma

1.73, s 1.03, s 0.96, d (6.5)

1.89, s

δC, type

δH (J in Hz)

79.3, CH 193.3, C

6.50, s

126.7, 161.1, 77.4, 31.6,

6.13, s

CH C C CH2

158.3, C 103.8, C 41.7, CH2 46.7, 126.1, 172.5, 8.6, 17.8, 19.7, 170.5, 20.5,

C C C CH3 CH3 CH3 C CH3

3.26, d (13.5) 3.13, d (13.5)

2.69, d (13.1) 2.75, d (13.1)

1.93, s 1.72, s 2.15, br s 2.13, s

4.86, d (7.2) 4.81, d (6.3) 7.12, s

7.68, s 9.61, br s

Overlapping signals. and 13C NMR data (see Table S1, Supporting Information); HRESIMS m/z 323.1489 [M − H]− (calcd for C17H23O6, 323.1500). Teuvislactone A (6): colorless oil, [α]25 D −96 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 215 (4.11) nm; ECD (MeOH, Δε) λmax 360 (−0.1), 306 (+0.5), 242 (−5.8) nm; IR (KBr) νmax 3735, 3396, 2921, 2852, 1733, 1692, 1437, 1258, 1201, 1130, 1028, 944, 801 cm−1; 1H and 13C NMR data (see Table 3); HRESIMS m/z 323.1503 [M − H]− (calcd for C17H23O6, 323.1500). Teuvislactone B (7): colorless, amorphous solid; [α]25 D −204 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 219 (4.15) nm; ECD (MeOH, Δε) λmax 344 (−0.2), 302 (+0.9), 242 (−13.0) nm; IR (KBr) νmax 3457, 3375, 3195, 2968, 2917, 1734, 1691, 1464, 1436, 1176, 1008, 770, 604 cm−1; 1H and 13C NMR data (see Table 3); HRESIMS m/z 565.3000 [2 M + H]+ (calcd for C30H45O10, 565.3007). (−)-(1S,5S,8S,10R)-1-Acetoxy-8-hydroxy-2-oxoeudesman-3,7(11)dien-8,12-olide (8): colorless block crystal (CH3OH); mp 206−207 °C; [α]25 D −110 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (3.80) nm; ECD (MeOH, Δε) λmax 320 (−0.7), 262 (−1.2), 238 (+4.5), 210 (−16.2) nm; IR (KBr) νmax 3408, 2976, 2918, 2891, 1745, 1675, 1376, 1227, 1091, 948, 839 cm−1; 1H and 13C NMR data (see Table S1, Supporting Information); HRESIMS m/z 321.1336 [M + H]+ (calcd for C17H21O6, 321.1338). (−)-(1S,5S,8S,10R)-1,8-Dihydroxy-2-oxoeudesman-3,7(11)-dien8,12-olide (9): colorless, amorphous solid; [α]25 D −182 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 223 (4.18) nm; ECD (MeOH, Δε) λmax 318 (−1.5), 264 (−1.4), 240 (+7.4), 212 (−19.7) nm; IR (KBr) νmax 3737, 3397, 2975, 1746, 1670, 1433, 1381, 1203, 1118, 1033, 944, 725 cm−1; 1 H and 13C NMR data (see Table S1, Supporting Information); HRESIMS m/z 277.1078 [M − H]− (calcd for C15H17O5, 277.1081). Teuvislactone C (10): colorless, amorphous solid; [α]25 D −74 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 223 (4.15) nm; ECD (MeOH, Δε) λmax 328 (−0.4), 272 (−0.1), 244 (+5.8), 212 (−18.2) nm; IR (KBr) νmax 3367, 2948, 1749, 1680, 1625, 1432, 1376, 1243, 1050, 765 cm−1; 1H and 13C NMR data (see Table 3); HRESIMS m/z 335.1127 [M − H]− (calcd for C17H19O7, 335.1136).

was obtained from fraction F5.4.3 (75.3 mg) by ODS (MeOH−H2O, 30−90%), and compound 10 (15.4 mg) was obtained from fraction F5.2 (336.7 mg) by ODS (MeOH−H2O, 30−80%) followed by further purification by preparative HPLC (CH3CN−H2O, 28%). 2α,11,12-Trihydroxy-7β,20-epoxy-8,11,13-abietatriene (1): yellow crystals (CH3OH−H2O, 10:1); mp 238−239 °C; [α]25 D −108 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.65) nm; IR (KBr) νmax 3339, 2956, 2928, 2871, 1739, 1585, 1441, 1366, 1228, 1217, 1052, 904, 528 cm−1; 1H and 13C NMR data (see Table 1); HRESIMS m/z 333.2068 [M + H]+ (calcd for C20H29O4, 333.2060). Teuvisone (2): yellow, amorphous solid; [α]25 D +6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (4.37) nm; IR (KBr) νmax 3411, 2958, 2872, 1712, 1675, 1655, 1573, 1365, 1221, 1048, 969, 671 cm−1; 1H and 13C NMR data (see Table 1); HRESIMS m/z 353.1722 [M + Na]+ (calcd for C20H26O4Na, 353.1723). Biteuvisone A (3): yellow, amorphous solid; [α]25 D −4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (4.70) nm; ECD (MeOH, Δε) λmax 368 (−0.6), 326 (+2.0), 286 (−3.7), 230 (+6.3), 220 (+5.3), 212 (+6.3) nm; IR (KBr) νmax 3335, 2956, 2871, 1738, 1650, 1438, 1367, 1228, 1217, 1046, 528 cm−1; 1H and 13C NMR data (see Table 2); HRESIMS m/z 695.3350 [M + Cl]− (calcd for C40H52O8Cl, 695.3356). Biteuvisone B (4): yellow, amorphous solid; [α]25 D −162 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.80) nm; ECD (MeOH, Δε) λmax 356 (+0.6), 316 (−1.4), 272 (+4.1), 228 (−11.7), 220 (−11.4), 214 (−11.7) nm; IR (KBr) νmax 3338, 2955, 2925, 2855, 1738, 1721, 1685, 1460, 1439, 1367, 1217, 1049, 800 cm−1; 1H and 13 C NMR data (see Table 2); HRESIMS m/z 1343.7202 [2 M + Na]+ (calcd for C80H104O16Na, 1343.7217). (1R,3S,4R,5R,8R,10S)-3-Acetoxy-1,8-dihydroxyeudesm-7(11)8,12-olide (5): colorless block crystals (CH3OH); mp 207−208 °C; [α]25 D −162 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (4.29) nm; ECD (MeOH, Δε) λmax 360 (−0.1), 302 (+0.8), 238 (−12.5) nm; IR (KBr) νmax 3368, 2966, 1737, 1374, 1251, 1025, 946, 767 cm−1; 1H G



Article

respectively. Copies of the data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk. Computational Work. Conformational analysis was performed with SYBYL-X V1.1.2 software using the MMFF94S force field with an energy cutoff of 10 kcal/mol. The obtained conformers were used for geometry reoptimizatons at the B3 LYP/6-31G(d) level with a PCM solvent model for methanol in the Gaussian 09 software.12 TDDFT ECD calculations for the optimized conformers were performed at the B3 LYP/6-31+G(d) level in methanol. ECD curves were obtained based on the rotatory strengths with a half-band of 0.3 eV using SpecDis v1.51.13 The ECD spectra and values were calculated from the spectra of individual conformers according to their contribution to the Boltzmann weighting after UV correction. Cytotoxicity Assay. Five human cancer cell lines, namely, H460, HepG2, BGC-823, HCT-116, and HeLa, were used to evaluate the cytotoxic effects of compounds 1−10 using the MTT method.14 The IC50 value is defined as the concentration of material required to reduce the absorbance of the control assay by 50%. Adriamycin was used as a positive control and gave IC50 values of 0.24 ± 0.02, 0.24 ± 0.07, 0.76 ± 0.10, 0.41 ± 0.13, and 0.72 ± 0.10 μM against the H460, HepG2, BGC-823, HCT-116, and HeLa cell lines, respectively. The results presented are the mean values of three independent experiments. α-Glucosidase Inhibitory Assay. Inhibitory α-glucosidase (from Saccharomyces cerevisiae; Sigma-Aldrich, St. Louis, MO, USA) activities were determined using p-nitrophenyl-α-D-glucopyranoside (PNPG) as the substrate, according to a reported method with minor modifications.15 Briefly, 100 μL of enzyme solution [1.0 U/mL αglucosidase in 0.1 M K3PO4 buffer (pH 6.9)] and 20 μL of the test compound in DMSO were mixed and preincubated for 10 min at 25 °C prior to initiating the reaction by the addition of the substrate. After preincubation, 50 μL of PNPG solution [5.0 mM PNPG in 0.1 M K3PO4 buffer (pH 6.9)] was added to each well of a 96-well plate, and the UV absorption of each well was measured immediately. After the measurement, the mixtures were incubated for 5 min at 25 °C, and their UV absorptions were measured again. The amount of PNPG released was quantified using a microplate reader (BioTek Synergy H1, BioTek Instruments, Inc.) at 405 nm. The increased absorbance (ΔA) was compared with that of the control (DMSO in place of the sample solution) to calculate the inhibition using the following equation:

Figure 7. Experimental ECD spectra of 5−10.

Inhibition (%) = (ΔAcontrol − ΔA sample)/ΔAcontrol × 100%

X-ray Crystallographic Analysis of 1. Yellow crystals of 1 were obtained from a 10:1 (v/v) mixture of MeOH and H2O using the vapor diffusion method. The structures were solved by direct methods using SHELXS-97 and were expanded using Fourier techniques (SHELXS-97). Crystal data of 1: C20H28O4 (M = 332.44); monoclinic crystal (0.46 × 0.50 × 0.63 mm); space group P212121; unit cell dimensions a = 10.734(7) Å, b = 12.344(3) Å, c = 13.282(5) Å, V = 1759(12) Å3; Z = 4; Dcalcd = 1.255 g/cm3, F(000) = 720, 3406 unique reflections [R(int) = 0.0509 (inf − 0.81 Å)], which were used in all of the calculations; the final refinement gave R1 = 0.0319 and wR2 = 0.0856 (w = 1/σ|F|2), S = 1.056; Flack parameter = −0.06(15). The X-ray crystallographic data of 5 are presented in the Supporting Information (S-1), and the crystallographic data for structures of 1 and 5 were deposited in the Cambridge Crystallographic Data Center with deposition numbers CCDC 1015116 (1) and CCDC 1025514 (5),

The concentration of inhibitor required to inhibit the α-glucosidase activity by 50% under the assay conditions was defined as the IC50 value. Acarbose was used as a positive control and gave an IC50 value of 169.3 μM. The results presented are the mean values of three independent experiments.

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ASSOCIATED CONTENT

S Supporting Information *

This material (1D and 2D NMR, HRESIMS, and IR spectra of compounds 1−4, 6, 7, and 10 and ECD computational protocol of compounds 3−5) is available free of charge via the Internet at http://pubs.acs.org.

Table 4. Cytotoxic Effects of Compounds 2 and 4a tumor cell growth inhibition (IC50, μM)

a

compound

H460

HepG2

BGC823

HCT116

HeLa

2 4 adriamycinb

>10 8.5 ± 1.10 0.24 ± 0.02

>10 5.2 ± 0.16 0.24 ± 0.07

>10 4.8 ± 0.60 0.76 ± 0.10

7.3 ± 1.74 6.3 ± 3.03 0.41 ± 0.13

>10 5.2 ± 1.04 0.72 ± 0.10

Compounds 1, 3, and 5−10 did not show cytotoxicity again these cell lines (IC50 > 10 μM). bPositive control for cytotoxic activities. H



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Article

Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision B.01; Gaussian, Inc.: Wallingford, CT, 2010. (13) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G. SpecDis, Version 1.51; University of Wuerzburg: Wuerzburg, Germany, 2011. (14) Zheng, D.; Han, L.; Li, Y. Q.; Li, J.; Rong, H.; Leng, Q.; Jiang, Y.; Zhao, L. X.; Huang, X. S. Molecules 2012, 17, 836−842. (15) Omar, R.; Li, L. Y.; Yuan, T.; Seeram, N. P. J. Nat. Prod. 2012, 75, 1505−1509.

AUTHOR INFORMATION

Corresponding Author

*Tel: +86-10-82805212. Fax: +86-10-82805212. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was funded by the National Natural Science Foundation of China (No. 81172943) and the National Key Scientific and Technological Special Projects (No. 2012ZX09103201-022).

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REFERENCES

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