Fitoterapia 98 (2014) 192–198
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Cytotoxic and anti-inflammatory ent-kaurane diterpenoids from Isodon wikstroemioides Hai-Yan Wu a,b, Wei-Guang Wang a, Hua-Yi Jiang a,b, Xue Du a, Xiao-Nian Li a, Jian-Xin Pu a,⁎, Han-Dong Sun a,⁎ a b
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e
i n f o
Article history: Received 27 June 2014 Accepted in revised form 4 August 2014 Available online 12 August 2014 Keywords: Lamiaceae Isodon wikstroemioides ent-Kaurane diterpenoid Cytotoxicity
a b s t r a c t Seven new ent-kaurane diterpenoids, isowikstroemins A–G (1–7), were isolated from EtOAc extracts of the aerial parts of Isodon wikstroemioides. Their structures were elucidated by extensive spectroscopic analysis. The isolates were evaluated for their cytotoxicity against five human tumor cell lines, and compounds 1–4 exhibited significant activity with IC50 values ranging from 0.9 to 7.0 μM. In addition, compounds 1, 2, 3, 4, and 7 exhibited inhibitory activity against nitric oxide (NO) production in LPS-activated RAW264.7 macrophages. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The genus Isodon (Lamiaceae) includes about 150 species and is distributed all over the world [1,2]. The use of Isodon species in Chinese folk medicines has a long tradition [3]. Over the past 30 years, phytochemical investigation of this genus has isolated and elucidated a large number of diterpenoids including ent-kaurane-type, abietane-type, isopimarane-type, gibberellane-type, labdane-type, and clerodane-type [4]. Many obtained diterpenoids exhibited interesting biological properties, such as antitumor, anti-inflammatory, and antibacterial activities [5–7]. Isodon wikstroemioides (Hand.-Mazz.) H. Hara, a perennial herb, is primarily distributed in the northwestern regions of Yunnan Province and the western district of Sichuan Province
⁎ Corresponding authors at: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, PR China. Tel.: +86 871 65223251. E-mail addresses: [email protected] (J.-X. Pu), [email protected] (H.-D. Sun).
http://dx.doi.org/10.1016/j.fitote.2014.08.012 0367-326X/© 2014 Elsevier B.V. All rights reserved.
in the People's Republic of China [8]. Previous phytochemical investigations of this plant have resulted in the isolation of 44 ent-kauranoids [9,10]. In our continuing work, seven new 7,20-epoxy-ent-kauranoids, isowikstroemins A–G (1–7), have been isolated from I. wikstroemioides. All of the isolates were evaluated for their cytotoxicity against the HL-60, SMMC-7721, A-549, MCF-7, and SW-480 human tumor cell lines, and tested for their ability to inhibit LPS-induced NO production in RAW264.7 macrophages. This paper reports the isolation, structure elucidation, and biological activities of these compounds.
2. Experimental 2.1. General experimental procedures Melting points of the isolates were obtained on an XRC-1 apparatus and were uncorrected. Optical rotations were measured in MeOH with Horiba SEPA-300 and JASCO P-1020 polarimeters. UV spectra were recorded using a Shimadzu UV2401A spectrophotometer. IR spectra were obtained on a Tenor 27 FT-IR spectrometer using KBr pellets. NMR spectra were
H.-Y. Wu et al. / Fitoterapia 98 (2014) 192–198
recorded on Bruker AM-400, DRX-500, and DRX-600 spectrometers using TMS as the internal standard. All chemical shifts (δ) are expressed in ppm relative to the solvent signals. HREIMS was performed on an API QSTAR TOF spectrometer. X-ray crystallographic data were collected on a Bruker APEX DUO diffractometer equipped with an APEX II CCD using Cu Kα radiation. Column chromatography (CC) was performed with 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 was performed on precoated TLC plates (200–250 μm thickness, silica gel 60 F254, Qingdao Marine Chemical, Inc.), and spots were visualized by UV light (254 nm) or by spraying heated silica gel plates with 10% H2SO4 in EtOH. Preparative HPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph with a Shimadzu PRC-ODS (K) column. Semi-preparative HPLC was performed on an Agilent 1100 liquid chromatograph with a ZORBAX SB-C18 (9.4 mm × 25 cm) column. 2.2. Plant material The aerial parts of I. wikstroemioides were collected in the Ranwu District of Sichuan Province, People's Republic of China, in July 2011 and identified by Prof. Xi-Wen Li at the Kunming Institute of Botany. A voucher specimen (KIB 20110939) has been deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences. 2.3. Extraction and isolation The dried and powdered aerial parts of I. wikstroemioides (7.5 kg) were extracted with 70% aqueous acetone (14 L) three times (three days each time) at room temperature and filtered. The filtrate was concentrated under reduced pressure and then partitioned between EtOAc and H2O. The EtOAc-soluble portion (380 g) was subjected to silica gel CC (100–200 mesh, 11 × 120 cm, 2 kg), eluted with CHCl3/acetone (1:0–0:1 gradient system) that afforded fractions A–G. The fractions were then decolorized using MCI gel and eluted with 90:10 MeOH/H2O. Fraction C (CHCl3/acetone, 8:2; 19 g), which was a brown gum, was subjected to RP-18 column chromatography (8 × 50 cm, MeOH/H2O 27:73 to 60:40 gradient) to provide three fractions, C1–C3. Fraction C2 (15 g) was separated into five subfractions (C2-1–C2-5) using RP-18 CC (6 × 40 cm, MeOH/H2O 25:75 to 40:60 gradient). C2-4 (9 g) was subjected to RP-18 CC (6 × 40 cm, CH3CN/H2O 35:65) to obtain 3 (4 g). C2-5 (5 g) was separated by preparative HPLC (6 × 29 cm, CH3CN/H2O 34:66) to afford 7 fractions (C2-5-1–C2-5-7). C2-5-6 (40 mg) was submitted to semi-preparative HPLC (5 μm, 9.4 × 250 mm, flow rate 3 ml/min, UV detection at λmax = 210, 254, and 280 nm, eluted with CH3CN/H2O 40:60, tR = 14 min) to yield 6 (4 mg). Compound 1 (2.4 mg) was isolated from fraction C2-5-7 (210 mg) by semi-preparative HPLC (MeOH/H2O 60:40, tR = 33 min). Fraction C3 (2 g) was separated by preparative HPLC (2.5 × 27 cm, CH3CN/H2O 34:66) to afford 17 fractions (C3-1–C3-17). C3-10 (105 mg) was submitted to semi-preparative HPLC (5 μm, 9.4 × 250 mm, flow rate 3 ml/min, UV detection at λmax = 210,
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254, and 280 nm, eluted with MeOH/CH3CN/H2O 15:30:55, tR = 7.7 min) to yield 4 (22 mg). C3-11 (65 mg) was submitted to semi-preparative HPLC (5 μm, 9.4 × 250 mm, flow rate 3 ml/min, UV detection at λmax = 210, 254, and 280 nm, eluted with MeOH/H2O 65:35, tR = 13.5 min) to yield 5 (11 mg). Fraction D (CHCl3/acetone, 7:3; 50 g), a brown gum, was subjected to silica gel CC (9 × 80 cm, 200–300 mesh, 1 kg), and eluted with CHCl3/MeOH (80:1) to afford seven fractions (D1–D7). D4 (20 g) was applied to a silica gel column (5 × 60 cm, 200–300 mesh, 200 g) and eluted with CHCl3/MeOH (80:1) to afford six fractions (D4-1–D4-6). D4-4 (14 g) was separated by preparative HPLC (6 × 29 cm, CH3CN/H2O 30:70) and then semi-preparative HPLC (9.4 × 250 mm, flow rate 3 ml/min, UV detection at λmax = 210, 254, and 280 nm, eluted with CH3CN/H2O 33:67, tR = 12.5 min) to yield 7 (3 mg). Compound 2 (29 mg) was obtained from fraction D4-5 (200 mg) by semi-preparative HPLC (9.4 × 250 mm, flow rate 3 ml/min, UV detection at λmax = 210, 254, and 280 nm, eluted with CH3CN/H2O 22:78, tR = 14 min). 2.4. Spectroscopic data Isowikstroemin A (1)colorless needles (MeOH); mp 136– 137 °C; [α]26 D : −60 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 231 (3.90), 196 (3.59) nm; IR (KBr) νmax 3446, 2927, 1726, 1646, 1235, 1027 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 427 [M + Na]+ (100); positive-ion HREIMS [M]+ m/z 404.2194 (calcd for C23H32O6, 404.2199). Isowikstroemin B (2)White amorphous powder; [α]26 D : −91 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 231 (3.86), 195 (3.55) nm; IR (KBr) νmax 3440, 2933, 1726, 1644, 1237, 1031 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 413 [M + Na]+ (100); positive-ion HREIMS [M]+ m/z 390.2049 (calcd for C22H30O6, 390.2042). Isowikstroemin C (3)White amorphous powder; [α]26 D : −51 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 230 (3.91), 196 (3.61) nm; IR (KBr) νmax 3432, 2926, 1722, 1646, 1269, 1056 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 385 [M + Na]+ (100); positive-ion HREIMS [M]+ m/z 362.2076 (calcd for C21H30O5, 362.2093). Isowikstroemin D (4)White amorphous powder; [α]26 D : −68 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 229 (3.86) nm; IR (KBr) νmax 3418, 2945, 1727, 1650, 1256, 1094 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 371 [M + Na]+ (100); positive-ion HREIMS [M]+ m/z 348.1938 (calcd for C20H28O5, 348.1937). Isowikstroemin E (5)White amorphous powder; [α]25 D : +4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (3.79) nm; IR (KBr) νmax 3441, 2928, 1719, 1659, 1242, 1029 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 429 [M + Na]+ (100); positive-ion HREIMS [M]+ m/z 406.2353 (calcd for C23H34O6, 406.2355). Isowikstroemin F (6)White amorphous powder; [α]26 D : −27 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.80) nm; IR (KBr) νmax 3438, 2933, 1718, 1630, 1246, 1030 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 415 [M + Na]+ (100); positive-ion HREIMS [M]+ m/z 392.2185 (calcd for C22H32O6, 392.2199). Isowikstroemin G (7)White amorphous powder; [α]26 D : −31 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.86) nm; IR (KBr)
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Table 1 1 H NMR data of compounds 1–7 (δ in ppm, J in Hz). Position
1a
2a
3b
4b
5a
6a
7a
1a 1b 2a 2b 3a 3b 5 6a 6b 7 9 11a 11b 12a 12b 13 14a 14b 15 17a 17b 18a 18b 19 20a 20b MeO AcO
2.00, overlap 0.82, m 1.51, overlap 1.42, overlap 1.70, m 1.51, overlap 1.96, overlap 3.14, overlap 1.87, overlap 4.30, d (2.6) 1.46, overlap 2.53, m 1.26, m 2.27, m 1.34, m 3.15, overlap 6.19, s
2.31, overlap 0.92, m 1.60, overlap 1.48, overlap 1.72, m 1.55, overlap 2.00, overlap 3.20, overlap 1.96, overlap 4.43, s 1.58, overlap 3.00, m 1.43, overlap 2.36, overlap 1.42, overlap 3.26, overlap 6.51, s
2.08, m 0.85, m 1.54, overlap 1.46, overlap 1.69, m 1.56, overlap 1.97, dd (10.9, 7.1) 3.25, m 1.91, m 4.72, d (2.2) 1.49, overlap 2.52, m 1.32, m 2.33, m 1.46, overlap 3.17, d (9.6) 5.14, s
2.40, overlap 0.96, m 1.62, overlap 1.49, overlap 1.73, overlap 1.59, overlap 2.01, overlap 3.31, overlap 2.00, overlap 4.84, s 1.60, overlap 3.01, m 1.48, overlap 2.38, overlap 1.51, overlap 3.20, d (9.5) 5.58, s
2.13, m 0.83, m 1.55, overlap 1.41, overlap 1.64, m 1.56, overlap 2.18, dd (11.0, 6.8) 2.66, t (12.3) 1.87, m 4.29, d (2.2) 2.35, overlap 2.47, m 1.23, m 2.32, overlap 1.48, overlap 2.82, d (9.1) 5.95, s
2.44, overlap 0.94, m 1.67, overlap 1.47, overlap 1.66, overlap 1.60, overlap 2.25, dd (10.9, 7.1) 2.72, t (12.2) 1.97, overlap 4.42, s 2.43, overlap 2.94, overlap 1.38, m 2.41, overlap 1.55, overlap 2.95, overlap 6.26, s
1.25, m 0.86, m 1.38, m
6.13, s 5.33, s 3.52, d (10.4) 3.34, d (10.4) 1.01, s 5.30, s
6.16, s 5.35, s 3.54, d (10.5) 3.37, d (10.5) 1.01, s 6.08, s
6.19, s 5.37, s 3.52, d (10.5) 3.36, d (10.5) 1.04, s 5.35, s
6.21, s 5.38, s 3.55, d (10.5) 3.39, d (10.5) 1.05, s 6.15, s
4.78, s 5.55, s 5.27, s 3.51, d (10.5) 3.37, d (10.5) 1.07, s 5.37, s
4.86, s 5.59, s 5.29, s 3.55, d (10.4) 3.42, d (10.4) 1.08, s 6.15, s
3.46, s 1.88, s
1.85, s
3.50, s 1.93, s
1.91, s
a b
3.41, s
1.60, m 2.32, dd (11.1, 7.1) 3.16, t (12.1) 2.13, overlap 2.22, m 1.51, overlap 1.14, m 2.12, overlap 1.50, overlap 2.63, m 2.01, dd (11.9, 4.6) 1.92, d (11.9) 5.07, s 5.50, s 5.23, s 3.52, d (10.4) 3.40, d (10.4) 1.20, s 4.36, d (9.3) 4.08, d (9.3)
Recorded at 400 MHz in pyridine-d5. Recorded at 500 MHz in pyridine-d5.
νmax 3392, 2928, 1635, 1388, 1130, 1032 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 357 [M + Na]+ (100); positive-ion HREIMS [M]+ m/z 334.2144 (calcd for C20H30O4, 334.2144).
2.5. X-ray crystal structure analysis The intensity data for isowikstroemin A (1), were collected at 100 K on a Bruker APEX DUO diffractometer equipped with
Table 2 13 C NMR data of compounds 1–7 (δ in ppm). Position
1a
2a
3b
4a
5b
6b
7a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 MeO AcO
29.9, t 18.0, t 34.8, t 38.9, s 42.1, d 25.2, t 66.2, d 56.6, s 50.5, d 39.2, s 19.6, t 31.6, t 40.9, d 75.2, d 204.7, s 152.1, s 116.6, t 70.6, t 17.0, q 102.2, d 55.6, q 20.8, q 170.2, s
30.6, t 18.1, t 35.0, t 39.3, s 42.3, d 25.5, t 66.1, d 57.0, s 51.1, d 39.1, s 19.8, t 31.9, t 40.9, d 76.0, d 204.9, s 152.4, s 116.7, t 70.9, t 17.1, q 94.5, d
30.4, t 18.1, t 35.1, t 39.1, s 42.4, d 25.7, t 66.9, d 58.7, s 50.7, d 39.4, s 19.9, t 31.9, t 43.8, d 70.7, d 206.4, s 154.5, s 115.6, t 70.9, t 17.2, q 102.5, d 55.7, q
31.0, t 18.2, t 35.2, t 39.2, s 42.5, d 26.0, t 66.8, d 59.2, s 51.1, d 39.5, s 20.1, t 32.1, t 43.9, d 71.0, d 206.7, s 154.8, s 115.5, t 71.1, t 17.2, q 94.6, d
30.7, t 18.3, t 35.4, t 38.8, s 42.4, d 26.6, t 69.9, d 51.3, s 43.7, d 39.0, s 18.0, t 33.3, t 43.0, d 77.5, d 74.9, d 162.4, s 108.8, t 71.3, t 17.2, q 102.9, d 55.6, q 21.1, q 170.4, s
31.3, t 18.4, t 35.6, t 38.8, s 42.4, d 27.0, t 69.7, d 51.5, s 44.2, d 39.1, s 18.2, t 33.6, t 42.9, d 78.2, d 75.0, d 162.7, s 108.9, t 71.6, t 17.3, q 95.1, d
31.7, t 18.8, t 35.8, t 39.1, s 42.8, d 34.1, t 96.7, s 52.6, s 43.3, d 35.0, s 15.6, t 33.2, t 36.4, d 26.3, t 75.9, d 164.7, s 107.1, t 71.4, t 17.4, q 67.0, t
a b
Recorded at 100 MHz in pyridine-d5. Recorded at 125 MHz in pyridine-d5.
20.9, q 170.3, s
21.1, q 170.5, s
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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 [11], expanded using difference Fournier techniques, and refined by the program and full-matrix least-squares calculations. The nonhydrogen 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 1007940 for 1. 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 isowikstroemin A (1): 2(C23H32O6)· C3H6O, M = 867.05, monoclinic, a = 14.0233(4) Å, b = 11.2595(3) Å, c = 27.8404(8) Å, α = 90.00°, β = 97.1520(10)°, γ = 90.00°, V = 4361.7(2) Å3, T = 100(2) K, space group C2, Z = 4, μ(Cu Kα) = 0.771 mm− 1, 20741 reflections measured, 7427 independent reflections (Rint = 0.0641). The final R1 values were 0.0844 (I N 2σ(I)). The final wR(F2) values were 0.2249 (I N 2σ(I)). The final R1 values were 0.0992 (all data). The final wR(F2) values were 0.2393 (all data). The goodness of fit on F2 was 1.042. Flack parameter = 0.1(2). The Hooft parameter is 0.05(11) for 3214 Bijvoet pairs. 2.6. Cytotoxicity assays The human tumor cell lines HL-60, SMMC-7721, A-549, MCF-7, and SW-480 were used, which were obtained from ATCC (Manassas, VA, USA). All the cells were cultured in RPMI1640 or DMEM medium (HyClone, Logan, UT, USA), supplemented with 10% fetal bovine serum (HyClone) at 37 °C in a humidified atmosphere with 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 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Sigma, St. Louis, MO, USA) [13]. Briefly, 100 μL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 × 105 cells/mL in 100 μL medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cis-platin and paclitaxel (Sigma) as positive controls. After the incubation, MTS (100 μg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20% SDS-50% DMF after removal of 100 μL medium. The optical density of the lysate was measured at 490 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by the Reed and Muench's method [14]. 2.7. Nitric oxide production in RAW264.7 macrophages Murine monocytic RAW264.7 macrophages were dispensed into 96-well plates (2 × 105 cells/well) containing RPMI 1640 medium (HyClone) with 10% FBS under a humidified atmosphere of 5% CO2 at 37 °C. After 24 h preincubation, cells were treated with serial dilutions of the compounds, with the maximum concentration of 25 μM, in the presence of 1 μg/mL
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LPS for 18 h. Each compound was dissolved in DMSO and further diluted in medium to produce different concentrations. NO production in each well was assessed by adding 100 μL of Griess reagent (Reagent A & Reagent B, respectively, Sigma) to 100 μL of each supernatant from LPS (Sigma)- treated or LPSand compound-treated cells in triplicate. After 5 min incubation, the absorbance was measured at 570 nm with a 2104 Envision Multilabel Plate Reader (PerkinElmer Life Sciences, Inc., Boston, MA, USA). MG-132 was used as a positive control [15]. 3. Results and discussion The air-dried and powdered aerial parts of I. wikstroemioides (7.5 kg) was extracted with 70% aqueous acetone and filtered. The filtrate was evaporated in vacuo. Then the concentrate was partitioned between EtOAc and H2O. The EtOAc-soluble partition (380 g) was subjected to column chromatography over silica gel, MCI CHP-20 gel, and Lichroprep RP-18, after which it was further purified by semi-preparative HPLC to afford seven new ent-kauranoids that have been named isowikstroemins A–G (1–7) (Fig. 1). Isowikstroemin A (1), colorless needles from MeOH, possessed a molecular formula of C23H32O6 with eight degrees of unsaturation, as established by HREIMS ([M]+ 404.2194, calcd 404.2199). Its IR absorption bands at 3446, 1726 and 1646 cm−1 indicated the presence of hydroxy, carbonyl, and alkenyl groups. Its 1H NMR data (Table 1) displayed characteristic resonances of a methyl (δH 1.01), a methoxy (δH 3.46), and an acetoxy (δH 1.88), while its 13C NMR and DEPT data (Table 2) exhibited 23 carbon resonances including an exocyclic double bond, an acetoxy group, a methoxy group, a carbonyl carbon, six methine carbons (of which three are oxygenated), seven methylene carbons (one of which is oxygenated), three quaternary carbons, and a methyl carbon. The proton and protonated carbon resonances in the NMR spectra of 1 were assigned by interpretation of 1H NMR, 13C NMR, and HSQC spectroscopic data. In the absence of any other sp or sp2 carbon, the above-mentioned analysis accounted for three out of eight degrees of unsaturation, indicating the structure of 1 must be pentacyclic diterpenoid. The 1H–1H COSY (Fig. 2) correlations of H2-1/H2-2/H2-3, H-5/H2-6/H-7, and H-9/H2-11/H2-12/H-13/H-14 were observed, together with HMBC correlations from H-5 to C-4, C-6, C-9, C-18, C-19, and C-20, from H-9 to C-8, C-11, C-14, C-15, and C-20, and from H-13 to C-8, C-11, C-12, C-14, C-15, and C-16 suggested that 1 is an ent-kaurane diterpenoid. Further analysis of the 2D NMR spectroscopic data of 1 revealed that 1 is a 7,20-epoxyent-kaurane diterpenoid with an acetal moiety at C-20. This assumption was confirmed by the HMBC correlations of H-1, H-5, H-7, and H-9 with C-20 (δC 102.2, d). The acetoxy group was located at C-14 as demonstrated by the HMBC correlations from H-14 (δH 6.19, s) to C-8, C-9, C-12, C-15, C-16, and acetyl carbonyl. C-18 was substituted with hydroxy group as suggested by the HMBC correlations of H2-18 (δH 3.52 and 3.34, 2H, each d, J = 10.4 Hz) with C-3, C-4, C-5, and C-19. HMBC correlations of H-20 (δH 5.30, s) with C-1, C-5, C-7, C-9, and the methoxy carbon indicated that the methoxy group was located at C-20. The relative configuration of 1 was determined via its ROESY spectrum (Fig. 2): the ROESY correlations of H2-18/H-5β
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Fig. 1. Chemical structures of compounds 1–7.
and of H-5β/H-9β indicated the β-orientation of H-5, H-9, and C-18, while the ROESY correlations of H-14/H-11α suggested the α-orientation of H-14. Moreover, the ROESY correlation of H-20/Me-19α revealed that the relative configuration of C-20 was S*, and this assignment was also supported by referring to isorosthins C and D [16]. The C-20R* of isorosthin C and C-20S* of isorosthin D were determined by the ROESY correlations of MeO-20/Me-19α and of H-20/Me-19α, respectively, which was confirmed by the single-crystal X-ray diffraction analysis of isorosthin C [16]. For the purpose of determining the absolute configuration of 1, we thus resorted to an X-ray diffraction experiment using the anomalous scattering of Cu Kα radiation yielded a Flack parameter of 0.1(2) [11] and a Hooft parameter of 0.05(11) for
1
H-1H COSY: H
H HMBC : H
3214 Bijvoet pairs, confirming the absolute configuration of 1 as 4S, 5R, 7R, 8R, 9R, 10S, 13S, 14R, and 20S (Fig. 3). Therefore, the structure of 1 was assigned as 20(S)-18-hydroxy-20methoxy-14β-acetoxy-7,20-epoxy-ent-kaur-16-en-15-one. The HREIMS ([M]+ 390.2049, calcd 390.2042) for isowikstroemin B (2) indicated a molecular formula of C22H30O6. The NMR data of 2 closely resemble those of compound 1, but for the downfield shift for H-20 from δH 5.30 in 1 to δH 6.08 in 2 and upfield shift for C-20 from δC 102.2 in 1 to δC 94.5 in 2, together with the absence of the methoxy group suggested that a hydroxy group instead of a methoxy group at C-20 in 2 [12]. This assumption was confirmed by the HMBC correlations of H-20 with C-1, C-5, C-7, C-9, and C-10. The ROESY spectrum of 2 indicated that the relative configurations
C ROESY : H
Fig. 2. 1H–1H COSY, selected HMBC, and key ROESY correlations of 1.
H
H.-Y. Wu et al. / Fitoterapia 98 (2014) 192–198
Fig. 3. The ORTEP drawing of compound 1.
of the stereogenic centers in 2 were identical to those of 1. Compound 2 was thus identified as 20(S*)-18,20-dihydroxy14β-acetoxy-7,20-epoxy-ent-kaur-16-en-15-one. The molecular formula of isowikstroemin C (3) was determined to be C21H30O5 by HREIMS ([M]+ 362.2076, calcd 362.2093), indicating seven degrees of unsaturation. Analysis of its 1D and 2D NMR data suggested that 3 is a 7,20-epoxy-entkauranoid with a methyl group, an olefinic group, a methoxy group, seven methylene (one oxygenated), six methine (three oxygenated), and four quaternary carbons (one carbonyl). The presence of a methoxy group at C-20 and two hydroxy groups at C-14 and C-18 were supported by the HMBC correlations of the protons of methoxy group (δH 3.41, s) with C-20, of H-14 (δH 5.14, s) with C-9, C-12, C-15, and C-16, and of H2-18 (δH 3.52 and 3.36, 2H, each d, J = 10.5 Hz) with C-3, C-4, C-5, and C-19. The relative configuration of 3 was determined by ROESY spectral data analysis. The correlation between H-14 and H-11α suggested the α-orientation of H-14, while the correlation between H2-18 and H-5β demonstrated the β-orientation of the C-18 hydroxymethyl group. Similarly, the correlation between H-20 and Me-19α indicated the relative configuration of C-20 as S*. Therefore, the structure of compound 3 was defined as 20(S*)-14β,18-dihydroxy-20-methoxy-7,20-epoxyent-kaur-16-en-15-one. Isowikstroemin D (4), was isolated as a white amorphous powder, and its molecular formula was established to be C20H28O5 by HREIMS ([M]+ 348.1938, calcd 348.1937). Comparisons of the 1H and 13C NMR data of 4 with those of 3 (Tables 1 and 2) indicated that both compounds have identical skeletons and substitution patterns, differing only in that 4 has a hydroxy group at C-20 rather than a methoxy group in 3, causing downfield shift for H-20 from δH 5.35 in 3 to δH 6.15 in 4 and upfield shift for C-20 from δC 102.5 in 3 to δC 94.6 in 4. This conclusion was verified by the HMBC correlations of H-20 with C-1, C-5, C-7, C-9, and C-10. The ROESY spectrum of 4 indicated that the relative configurations of its stereogenic carbons were identical to those of 3. Compound 4 was thus identified as 20(S*)-14β,18,20-trihydroxy-7,20-epoxy-ent-kaur16-en-15-one.
197
Isowikstroemin E (5) has a molecular formula of C23H34O6 based on HREIMS ([M]+ 406.2353, calcd 406.2355), indicating seven degrees of unsaturation. Except for one acetoxy group, 21 carbon atoms found in the 13C NMR and DEPT spectra consisted of an olefinic quaternary carbon, an olefinic methylene carbon, a methoxy carbon, seven methylene carbons (one oxygenated), seven methine carbons (four oxygenated), three quaternary carbons, and a methyl carbon, which were again consistent with a skeleton of a 7,20-epoxy-ent-kaurane diterpenoid. Two hydroxy groups at C-15 and C-18 based on the HMBC correlations of H-15 (δH 4.78, s) with C-7, C-9, and C-16, and of H2-18 (δH 3.51 and 3.37, 2H, each d, J = 10.5 Hz) with C-3, C-4, C-5, and C-19. A methoxy group and an acetoxy group located at C-20 and C-14, respectively, according to the HMBC correlations from H-20 (δH 5.37, s) to C-1, C-7, C-9, C-10, and methoxy carbon, and from H-14 (δH 5.95, s) to C-8, C-9, C-12, C-16, and acetyl carbonyl. A ROESY experiment revealed that H-14 and H-15 in 5 were both α-oriented based on the correlations of H-14/H-11α, and of H-15/H-13α, while C-18 was β-oriented based on the correlation of H2-18/H-5β. The relative configuration of C-20 was assigned as S* according to the ROESY correlation of H-20 and Me-19α. Therefore, the structure of 5 was defined as 20(S*)-15β,18-dihydroxy-20methoxy-14β-acetoxy-7,20-epoxy-ent-kaur-16-ene. The HREIMS ([M]+ 392.2185, calcd 392.2199) for isowikstroemin F (6) indicated a molecular formula of C22H32O6. The 1H and 13C NMR data of 6 were similar to those of 5 (Tables 1 and 2) except that the hydroxy group at C-20 in 6 instead of the methoxy group in 5. Downfield shift for H-20 from δH 5.37 in 5 to δH 6.15 in 6 and upfield shift for C-20 from δC 102.9 in 5 to δC 95.1 in 6, together with the HMBC correlations of H-20 with C-5, C-7, C-9, and C-10 confirmed this assumption. In addition, correlations observed in the ROESY spectrum indicated that the orientations of the substituents in 6 were the same as in 5. The structure of compound 6 was thus defined as 20(S*)-15β, 18, 20-trihydroxy-14βacetoxy-7,20-epoxy-ent-kaur-16-ene. The molecular formula of isowikstroemin G (7) was determined to be C20H30O4 by HREIMS ([M]+ 334.2144, calcd 334.2144), indicating six degrees of unsaturation. Its IR absorption bands at 3392 and 1635 cm−1 indicated the presence of hydroxy and alkenyl groups. Its 13C NMR and DEPT data (Table 2) exhibited 20 carbon resonances including an olefinic group, a methyl group, nine methylene (two of which are oxygenated), four methine (one of which is oxygenated), and four quaternary carbons (which one is oxygenated). Protons were assigned to related carbon resonances via the 1H NMR, 13C NMR, and HSQC spectroscopic data. Based on its NMR data, compound 7 is also a 7,20-epoxy-ent-kaurane diterpenoid. The presence of two hydroxy groups at C-15 and C-18 in 7 was confirmed by the observation of HMBC correlations from H-9, H-13, H-14, and H-17 to C-15 (δC 75.9, d), and from H2-18 (δH 3.52 and 3.40, 2H, each d, J = 10.4 Hz) to C-3, C-4, C-5, and C-19. In addition, HMBC correlations of H2-20 (δH 4.36 and 4.08, 2H, each d, J = 9.3 Hz) with C-5, C-7 (δC 96.7, s), C-9, and C-10 suggested that C-20 connects with C-7 through an oxygen atom and forms the structure of hemiacetal at C-7. The ROESY correlation from H-15 to H-13α indicated that the C-15 hydroxy group in 7 was β-oriented. Therefore, the structure of compound 7 was assigned as 7β,15β,18-trihydroxy-7,20epoxy-ent-kaur-16-ene.
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Table 3 IC50 values (μM) of diterpenoids from I. wikstroemioides against Tumor Cell Lines.a Compound
HL-60
SMMC-7721
A-549
MCF-7
SW-480
1 2 3 4 5 6 7 DDPb Paclitaxelb
2.51 ± 0.16 2.48 ± 0.11 3.79 ± 0.06 2.97 ± 0.14 N40 N10 ± 1.22 N10 ± 0.54 1.75 ± 0.04 b0.008
1.02 ± 0.02 3.78 ± 0.02 4.72 ± 0.09 3.65 ± 0.07 N40 N10 ± 0.16 N10 ± 0.32 4.46 ± 0.08 b0.008
0.88 ± 0.04 3.34 ± 0.04 7.00 ± 0.20 3.84 ± 0.13 N40 N10 ± 1.02 N10 ± 0.75 7.61 ± 0.22 b0.008
2.1 ± 0.10 2.28 ± 0.07 5.35 ± 0.28 3.37 ± 0.06 N40 N40 4.79 ± 0.21 15.7 ± 0.08 b0.008
1.22 ± 0.08 2.40 ± 0.15 4.23 ± 0.11 2.51 ± 0.13 N40 N10 ± 0.48 4.23 ± 0.01 15.0 ± 0.03 b0.008
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. Each value represents the mean ± SEM (n = 3). b DDP (cis-platin) and paclitaxel were used as positive controls.
Table 4 IC50 values (μM) of diterpenoids from I. wikstroemioides against LPS-activated NO production in RAW264.7 cells.a
Appendix A. Supplementary data
Compound
IC50 (μM)
Compound
IC50 (μM)
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.08.012.
1 3 6 MG-132
1.34 1.49 23.6 0.13
2 4 7
2.28 ± 0.17 1.13 ± 0.14 2.50 ± 0.37
References
± ± ± ±
0.11 0.08 0.41 0.07
a Each value represents the mean ± SEM (n = 3). Compounds with values of IC50 b 10 µM exhibited interesting inhibitory activity against NO production.
All of the isolates obtained from I. wikstroemioides in this work were evaluated for their cytotoxicity against the HL-60 (acute leukemia), SMMC-7721 (hepatic cancer), A-549 (lung cancer), MCF-7 (breast cancer), and SW-480 (colon cancer) human tumor cell lines using the MTS method [13], with cisplatin and paclitaxel as positive controls. Compounds 1–4 exhibited significant cytotoxic activity with IC50 values ranging from 0.9 to 7.0 μM, while compound 7 showed selective antiproliferative activity against the MCF-7 and SW-480 cancer cell lines, with IC50 values of 4.8 μM and 4.2 μM, respectively (Table 3). In addition, six isolates were tested for their capacity to inhibit NO production in LPS-stimulated RAW264.7 cells. Interesting inhibitory activity was observed for compounds 1, 2, 3, 4, and 7 with IC50 values ranging from 1.1 to 2.5 μM (Table 4). 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), the Major Direction Projection Foundation of CAS Intellectual Innovation Project (Grant KSCX2-EW-J-24), and the West Light Foundation of the Chinese Academy of Sciences (J.-X. P.).
[1] Flora Reipublicae Popularis Sinicae Tomus. Beijing: Science Press; 1977 428. [2] Flora Yunnanica Tomus. Beijing: Science Press; 1977 758. [3] The pharmacopoecia of People's Republic of China. Beijing: People's Health Press; 1977. [4] Sun HD, Huang SX, Han QB. Diterpenoids from Isodon species and their biological activities. Nat Prod Rep 2006;23:673–98. [5] Fujita E, Node M. In: Herz W, Grisebach H, Kirby GW, Tamm Ch, editors. Progress in the chemistry of organic natural products. Vienna: Springer Verlag; 1984. p. 77–157. [6] Takeda Y, Otsuka H. In: Atta-ur-Rahman, editor. Studies in natural products chemistry. Amsterdam: Elsevier; 1995. p. 111–85. [7] Sun HD, Xu YL, Jiang B. Diterpenoids from Isodon species. 1st ed. Beijing: Science Press; 2001 93. [8] Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita. Flora Reipublicae Popularis Sinica, 66. Beijing: Science Press; 1977 454. [9] Wu SH, Zhang HJ, Chen YP, Lin ZW, Sun HD. Diterpenoids from Isodon wikstroemioides. Phytochemistry 1993;34:1099–102. [10] Wu HY, Zhan R, Wang WG, Jiang HY, Pu JX, Sun HD, et al. Cytotoxic entkaurane diterpenoids from Isodon wikstroemioides. J Nat Prod 2014;77: 931–41. [11] Flack HD. On enantiomorph-polarity estimation. Acta Crystallogr A 1983; 39:876–81. [12] Huang H, Xu YL, Sun HD. Diterpenoids from Rabdosia coetsoides. Phytochemistry 1989;28:2753–7. [13] Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 1991;83:757–66. [14] Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. Am J Hyg 1938;27:493–7. [15] Fan JT, Su J, Peng YM. Rubiyunnanins C–H, cytotoxic cyclic hexapeptides from Rubia yunnanensis inhibiting nitric oxide production and NF-κB activation. Bioorg Med Chem 2010;18:8226–34. [16] Zhan R, Li XN, Du X, Wang WG, Pu JX, Sun HD, et al. Bioactive ent-kaurane diterpenoids from Isodon rosthornii. J Nat Prod 2013;76:1267–77.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Cytotoxic and anti-inflammatory ent-kaurane diterpenoids from Isodon wikstroemioides Hai-Yan Wu a,b, Wei-Guang Wang a, Hua-Yi Jiang a,b, Xue Du a, Xiao-Nian Li a, Jian-Xin Pu a,⁎, Han-Dong Sun a,⁎ a b
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e
i n f o
Article history: Received 27 June 2014 Accepted in revised form 4 August 2014 Available online 12 August 2014 Keywords: Lamiaceae Isodon wikstroemioides ent-Kaurane diterpenoid Cytotoxicity
a b s t r a c t Seven new ent-kaurane diterpenoids, isowikstroemins A–G (1–7), were isolated from EtOAc extracts of the aerial parts of Isodon wikstroemioides. Their structures were elucidated by extensive spectroscopic analysis. The isolates were evaluated for their cytotoxicity against five human tumor cell lines, and compounds 1–4 exhibited significant activity with IC50 values ranging from 0.9 to 7.0 μM. In addition, compounds 1, 2, 3, 4, and 7 exhibited inhibitory activity against nitric oxide (NO) production in LPS-activated RAW264.7 macrophages. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The genus Isodon (Lamiaceae) includes about 150 species and is distributed all over the world [1,2]. The use of Isodon species in Chinese folk medicines has a long tradition [3]. Over the past 30 years, phytochemical investigation of this genus has isolated and elucidated a large number of diterpenoids including ent-kaurane-type, abietane-type, isopimarane-type, gibberellane-type, labdane-type, and clerodane-type [4]. Many obtained diterpenoids exhibited interesting biological properties, such as antitumor, anti-inflammatory, and antibacterial activities [5–7]. Isodon wikstroemioides (Hand.-Mazz.) H. Hara, a perennial herb, is primarily distributed in the northwestern regions of Yunnan Province and the western district of Sichuan Province
⁎ Corresponding authors at: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, PR China. Tel.: +86 871 65223251. E-mail addresses: [email protected] (J.-X. Pu), [email protected] (H.-D. Sun).
http://dx.doi.org/10.1016/j.fitote.2014.08.012 0367-326X/© 2014 Elsevier B.V. All rights reserved.
in the People's Republic of China [8]. Previous phytochemical investigations of this plant have resulted in the isolation of 44 ent-kauranoids [9,10]. In our continuing work, seven new 7,20-epoxy-ent-kauranoids, isowikstroemins A–G (1–7), have been isolated from I. wikstroemioides. All of the isolates were evaluated for their cytotoxicity against the HL-60, SMMC-7721, A-549, MCF-7, and SW-480 human tumor cell lines, and tested for their ability to inhibit LPS-induced NO production in RAW264.7 macrophages. This paper reports the isolation, structure elucidation, and biological activities of these compounds.
2. Experimental 2.1. General experimental procedures Melting points of the isolates were obtained on an XRC-1 apparatus and were uncorrected. Optical rotations were measured in MeOH with Horiba SEPA-300 and JASCO P-1020 polarimeters. UV spectra were recorded using a Shimadzu UV2401A spectrophotometer. IR spectra were obtained on a Tenor 27 FT-IR spectrometer using KBr pellets. NMR spectra were
H.-Y. Wu et al. / Fitoterapia 98 (2014) 192–198
recorded on Bruker AM-400, DRX-500, and DRX-600 spectrometers using TMS as the internal standard. All chemical shifts (δ) are expressed in ppm relative to the solvent signals. HREIMS was performed on an API QSTAR TOF spectrometer. X-ray crystallographic data were collected on a Bruker APEX DUO diffractometer equipped with an APEX II CCD using Cu Kα radiation. Column chromatography (CC) was performed with 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 was performed on precoated TLC plates (200–250 μm thickness, silica gel 60 F254, Qingdao Marine Chemical, Inc.), and spots were visualized by UV light (254 nm) or by spraying heated silica gel plates with 10% H2SO4 in EtOH. Preparative HPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph with a Shimadzu PRC-ODS (K) column. Semi-preparative HPLC was performed on an Agilent 1100 liquid chromatograph with a ZORBAX SB-C18 (9.4 mm × 25 cm) column. 2.2. Plant material The aerial parts of I. wikstroemioides were collected in the Ranwu District of Sichuan Province, People's Republic of China, in July 2011 and identified by Prof. Xi-Wen Li at the Kunming Institute of Botany. A voucher specimen (KIB 20110939) has been deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences. 2.3. Extraction and isolation The dried and powdered aerial parts of I. wikstroemioides (7.5 kg) were extracted with 70% aqueous acetone (14 L) three times (three days each time) at room temperature and filtered. The filtrate was concentrated under reduced pressure and then partitioned between EtOAc and H2O. The EtOAc-soluble portion (380 g) was subjected to silica gel CC (100–200 mesh, 11 × 120 cm, 2 kg), eluted with CHCl3/acetone (1:0–0:1 gradient system) that afforded fractions A–G. The fractions were then decolorized using MCI gel and eluted with 90:10 MeOH/H2O. Fraction C (CHCl3/acetone, 8:2; 19 g), which was a brown gum, was subjected to RP-18 column chromatography (8 × 50 cm, MeOH/H2O 27:73 to 60:40 gradient) to provide three fractions, C1–C3. Fraction C2 (15 g) was separated into five subfractions (C2-1–C2-5) using RP-18 CC (6 × 40 cm, MeOH/H2O 25:75 to 40:60 gradient). C2-4 (9 g) was subjected to RP-18 CC (6 × 40 cm, CH3CN/H2O 35:65) to obtain 3 (4 g). C2-5 (5 g) was separated by preparative HPLC (6 × 29 cm, CH3CN/H2O 34:66) to afford 7 fractions (C2-5-1–C2-5-7). C2-5-6 (40 mg) was submitted to semi-preparative HPLC (5 μm, 9.4 × 250 mm, flow rate 3 ml/min, UV detection at λmax = 210, 254, and 280 nm, eluted with CH3CN/H2O 40:60, tR = 14 min) to yield 6 (4 mg). Compound 1 (2.4 mg) was isolated from fraction C2-5-7 (210 mg) by semi-preparative HPLC (MeOH/H2O 60:40, tR = 33 min). Fraction C3 (2 g) was separated by preparative HPLC (2.5 × 27 cm, CH3CN/H2O 34:66) to afford 17 fractions (C3-1–C3-17). C3-10 (105 mg) was submitted to semi-preparative HPLC (5 μm, 9.4 × 250 mm, flow rate 3 ml/min, UV detection at λmax = 210,
193
254, and 280 nm, eluted with MeOH/CH3CN/H2O 15:30:55, tR = 7.7 min) to yield 4 (22 mg). C3-11 (65 mg) was submitted to semi-preparative HPLC (5 μm, 9.4 × 250 mm, flow rate 3 ml/min, UV detection at λmax = 210, 254, and 280 nm, eluted with MeOH/H2O 65:35, tR = 13.5 min) to yield 5 (11 mg). Fraction D (CHCl3/acetone, 7:3; 50 g), a brown gum, was subjected to silica gel CC (9 × 80 cm, 200–300 mesh, 1 kg), and eluted with CHCl3/MeOH (80:1) to afford seven fractions (D1–D7). D4 (20 g) was applied to a silica gel column (5 × 60 cm, 200–300 mesh, 200 g) and eluted with CHCl3/MeOH (80:1) to afford six fractions (D4-1–D4-6). D4-4 (14 g) was separated by preparative HPLC (6 × 29 cm, CH3CN/H2O 30:70) and then semi-preparative HPLC (9.4 × 250 mm, flow rate 3 ml/min, UV detection at λmax = 210, 254, and 280 nm, eluted with CH3CN/H2O 33:67, tR = 12.5 min) to yield 7 (3 mg). Compound 2 (29 mg) was obtained from fraction D4-5 (200 mg) by semi-preparative HPLC (9.4 × 250 mm, flow rate 3 ml/min, UV detection at λmax = 210, 254, and 280 nm, eluted with CH3CN/H2O 22:78, tR = 14 min). 2.4. Spectroscopic data Isowikstroemin A (1)colorless needles (MeOH); mp 136– 137 °C; [α]26 D : −60 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 231 (3.90), 196 (3.59) nm; IR (KBr) νmax 3446, 2927, 1726, 1646, 1235, 1027 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 427 [M + Na]+ (100); positive-ion HREIMS [M]+ m/z 404.2194 (calcd for C23H32O6, 404.2199). Isowikstroemin B (2)White amorphous powder; [α]26 D : −91 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 231 (3.86), 195 (3.55) nm; IR (KBr) νmax 3440, 2933, 1726, 1644, 1237, 1031 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 413 [M + Na]+ (100); positive-ion HREIMS [M]+ m/z 390.2049 (calcd for C22H30O6, 390.2042). Isowikstroemin C (3)White amorphous powder; [α]26 D : −51 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 230 (3.91), 196 (3.61) nm; IR (KBr) νmax 3432, 2926, 1722, 1646, 1269, 1056 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 385 [M + Na]+ (100); positive-ion HREIMS [M]+ m/z 362.2076 (calcd for C21H30O5, 362.2093). Isowikstroemin D (4)White amorphous powder; [α]26 D : −68 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 229 (3.86) nm; IR (KBr) νmax 3418, 2945, 1727, 1650, 1256, 1094 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 371 [M + Na]+ (100); positive-ion HREIMS [M]+ m/z 348.1938 (calcd for C20H28O5, 348.1937). Isowikstroemin E (5)White amorphous powder; [α]25 D : +4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (3.79) nm; IR (KBr) νmax 3441, 2928, 1719, 1659, 1242, 1029 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 429 [M + Na]+ (100); positive-ion HREIMS [M]+ m/z 406.2353 (calcd for C23H34O6, 406.2355). Isowikstroemin F (6)White amorphous powder; [α]26 D : −27 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.80) nm; IR (KBr) νmax 3438, 2933, 1718, 1630, 1246, 1030 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 415 [M + Na]+ (100); positive-ion HREIMS [M]+ m/z 392.2185 (calcd for C22H32O6, 392.2199). Isowikstroemin G (7)White amorphous powder; [α]26 D : −31 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.86) nm; IR (KBr)
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H.-Y. Wu et al. / Fitoterapia 98 (2014) 192–198
Table 1 1 H NMR data of compounds 1–7 (δ in ppm, J in Hz). Position
1a
2a
3b
4b
5a
6a
7a
1a 1b 2a 2b 3a 3b 5 6a 6b 7 9 11a 11b 12a 12b 13 14a 14b 15 17a 17b 18a 18b 19 20a 20b MeO AcO
2.00, overlap 0.82, m 1.51, overlap 1.42, overlap 1.70, m 1.51, overlap 1.96, overlap 3.14, overlap 1.87, overlap 4.30, d (2.6) 1.46, overlap 2.53, m 1.26, m 2.27, m 1.34, m 3.15, overlap 6.19, s
2.31, overlap 0.92, m 1.60, overlap 1.48, overlap 1.72, m 1.55, overlap 2.00, overlap 3.20, overlap 1.96, overlap 4.43, s 1.58, overlap 3.00, m 1.43, overlap 2.36, overlap 1.42, overlap 3.26, overlap 6.51, s
2.08, m 0.85, m 1.54, overlap 1.46, overlap 1.69, m 1.56, overlap 1.97, dd (10.9, 7.1) 3.25, m 1.91, m 4.72, d (2.2) 1.49, overlap 2.52, m 1.32, m 2.33, m 1.46, overlap 3.17, d (9.6) 5.14, s
2.40, overlap 0.96, m 1.62, overlap 1.49, overlap 1.73, overlap 1.59, overlap 2.01, overlap 3.31, overlap 2.00, overlap 4.84, s 1.60, overlap 3.01, m 1.48, overlap 2.38, overlap 1.51, overlap 3.20, d (9.5) 5.58, s
2.13, m 0.83, m 1.55, overlap 1.41, overlap 1.64, m 1.56, overlap 2.18, dd (11.0, 6.8) 2.66, t (12.3) 1.87, m 4.29, d (2.2) 2.35, overlap 2.47, m 1.23, m 2.32, overlap 1.48, overlap 2.82, d (9.1) 5.95, s
2.44, overlap 0.94, m 1.67, overlap 1.47, overlap 1.66, overlap 1.60, overlap 2.25, dd (10.9, 7.1) 2.72, t (12.2) 1.97, overlap 4.42, s 2.43, overlap 2.94, overlap 1.38, m 2.41, overlap 1.55, overlap 2.95, overlap 6.26, s
1.25, m 0.86, m 1.38, m
6.13, s 5.33, s 3.52, d (10.4) 3.34, d (10.4) 1.01, s 5.30, s
6.16, s 5.35, s 3.54, d (10.5) 3.37, d (10.5) 1.01, s 6.08, s
6.19, s 5.37, s 3.52, d (10.5) 3.36, d (10.5) 1.04, s 5.35, s
6.21, s 5.38, s 3.55, d (10.5) 3.39, d (10.5) 1.05, s 6.15, s
4.78, s 5.55, s 5.27, s 3.51, d (10.5) 3.37, d (10.5) 1.07, s 5.37, s
4.86, s 5.59, s 5.29, s 3.55, d (10.4) 3.42, d (10.4) 1.08, s 6.15, s
3.46, s 1.88, s
1.85, s
3.50, s 1.93, s
1.91, s
a b
3.41, s
1.60, m 2.32, dd (11.1, 7.1) 3.16, t (12.1) 2.13, overlap 2.22, m 1.51, overlap 1.14, m 2.12, overlap 1.50, overlap 2.63, m 2.01, dd (11.9, 4.6) 1.92, d (11.9) 5.07, s 5.50, s 5.23, s 3.52, d (10.4) 3.40, d (10.4) 1.20, s 4.36, d (9.3) 4.08, d (9.3)
Recorded at 400 MHz in pyridine-d5. Recorded at 500 MHz in pyridine-d5.
νmax 3392, 2928, 1635, 1388, 1130, 1032 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS: m/z 357 [M + Na]+ (100); positive-ion HREIMS [M]+ m/z 334.2144 (calcd for C20H30O4, 334.2144).
2.5. X-ray crystal structure analysis The intensity data for isowikstroemin A (1), were collected at 100 K on a Bruker APEX DUO diffractometer equipped with
Table 2 13 C NMR data of compounds 1–7 (δ in ppm). Position
1a
2a
3b
4a
5b
6b
7a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 MeO AcO
29.9, t 18.0, t 34.8, t 38.9, s 42.1, d 25.2, t 66.2, d 56.6, s 50.5, d 39.2, s 19.6, t 31.6, t 40.9, d 75.2, d 204.7, s 152.1, s 116.6, t 70.6, t 17.0, q 102.2, d 55.6, q 20.8, q 170.2, s
30.6, t 18.1, t 35.0, t 39.3, s 42.3, d 25.5, t 66.1, d 57.0, s 51.1, d 39.1, s 19.8, t 31.9, t 40.9, d 76.0, d 204.9, s 152.4, s 116.7, t 70.9, t 17.1, q 94.5, d
30.4, t 18.1, t 35.1, t 39.1, s 42.4, d 25.7, t 66.9, d 58.7, s 50.7, d 39.4, s 19.9, t 31.9, t 43.8, d 70.7, d 206.4, s 154.5, s 115.6, t 70.9, t 17.2, q 102.5, d 55.7, q
31.0, t 18.2, t 35.2, t 39.2, s 42.5, d 26.0, t 66.8, d 59.2, s 51.1, d 39.5, s 20.1, t 32.1, t 43.9, d 71.0, d 206.7, s 154.8, s 115.5, t 71.1, t 17.2, q 94.6, d
30.7, t 18.3, t 35.4, t 38.8, s 42.4, d 26.6, t 69.9, d 51.3, s 43.7, d 39.0, s 18.0, t 33.3, t 43.0, d 77.5, d 74.9, d 162.4, s 108.8, t 71.3, t 17.2, q 102.9, d 55.6, q 21.1, q 170.4, s
31.3, t 18.4, t 35.6, t 38.8, s 42.4, d 27.0, t 69.7, d 51.5, s 44.2, d 39.1, s 18.2, t 33.6, t 42.9, d 78.2, d 75.0, d 162.7, s 108.9, t 71.6, t 17.3, q 95.1, d
31.7, t 18.8, t 35.8, t 39.1, s 42.8, d 34.1, t 96.7, s 52.6, s 43.3, d 35.0, s 15.6, t 33.2, t 36.4, d 26.3, t 75.9, d 164.7, s 107.1, t 71.4, t 17.4, q 67.0, t
a b
Recorded at 100 MHz in pyridine-d5. Recorded at 125 MHz in pyridine-d5.
20.9, q 170.3, s
21.1, q 170.5, s
H.-Y. Wu et al. / Fitoterapia 98 (2014) 192–198
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 [11], expanded using difference Fournier techniques, and refined by the program and full-matrix least-squares calculations. The nonhydrogen 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 1007940 for 1. 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 isowikstroemin A (1): 2(C23H32O6)· C3H6O, M = 867.05, monoclinic, a = 14.0233(4) Å, b = 11.2595(3) Å, c = 27.8404(8) Å, α = 90.00°, β = 97.1520(10)°, γ = 90.00°, V = 4361.7(2) Å3, T = 100(2) K, space group C2, Z = 4, μ(Cu Kα) = 0.771 mm− 1, 20741 reflections measured, 7427 independent reflections (Rint = 0.0641). The final R1 values were 0.0844 (I N 2σ(I)). The final wR(F2) values were 0.2249 (I N 2σ(I)). The final R1 values were 0.0992 (all data). The final wR(F2) values were 0.2393 (all data). The goodness of fit on F2 was 1.042. Flack parameter = 0.1(2). The Hooft parameter is 0.05(11) for 3214 Bijvoet pairs. 2.6. Cytotoxicity assays The human tumor cell lines HL-60, SMMC-7721, A-549, MCF-7, and SW-480 were used, which were obtained from ATCC (Manassas, VA, USA). All the cells were cultured in RPMI1640 or DMEM medium (HyClone, Logan, UT, USA), supplemented with 10% fetal bovine serum (HyClone) at 37 °C in a humidified atmosphere with 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 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Sigma, St. Louis, MO, USA) [13]. Briefly, 100 μL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 × 105 cells/mL in 100 μL medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cis-platin and paclitaxel (Sigma) as positive controls. After the incubation, MTS (100 μg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20% SDS-50% DMF after removal of 100 μL medium. The optical density of the lysate was measured at 490 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by the Reed and Muench's method [14]. 2.7. Nitric oxide production in RAW264.7 macrophages Murine monocytic RAW264.7 macrophages were dispensed into 96-well plates (2 × 105 cells/well) containing RPMI 1640 medium (HyClone) with 10% FBS under a humidified atmosphere of 5% CO2 at 37 °C. After 24 h preincubation, cells were treated with serial dilutions of the compounds, with the maximum concentration of 25 μM, in the presence of 1 μg/mL
195
LPS for 18 h. Each compound was dissolved in DMSO and further diluted in medium to produce different concentrations. NO production in each well was assessed by adding 100 μL of Griess reagent (Reagent A & Reagent B, respectively, Sigma) to 100 μL of each supernatant from LPS (Sigma)- treated or LPSand compound-treated cells in triplicate. After 5 min incubation, the absorbance was measured at 570 nm with a 2104 Envision Multilabel Plate Reader (PerkinElmer Life Sciences, Inc., Boston, MA, USA). MG-132 was used as a positive control [15]. 3. Results and discussion The air-dried and powdered aerial parts of I. wikstroemioides (7.5 kg) was extracted with 70% aqueous acetone and filtered. The filtrate was evaporated in vacuo. Then the concentrate was partitioned between EtOAc and H2O. The EtOAc-soluble partition (380 g) was subjected to column chromatography over silica gel, MCI CHP-20 gel, and Lichroprep RP-18, after which it was further purified by semi-preparative HPLC to afford seven new ent-kauranoids that have been named isowikstroemins A–G (1–7) (Fig. 1). Isowikstroemin A (1), colorless needles from MeOH, possessed a molecular formula of C23H32O6 with eight degrees of unsaturation, as established by HREIMS ([M]+ 404.2194, calcd 404.2199). Its IR absorption bands at 3446, 1726 and 1646 cm−1 indicated the presence of hydroxy, carbonyl, and alkenyl groups. Its 1H NMR data (Table 1) displayed characteristic resonances of a methyl (δH 1.01), a methoxy (δH 3.46), and an acetoxy (δH 1.88), while its 13C NMR and DEPT data (Table 2) exhibited 23 carbon resonances including an exocyclic double bond, an acetoxy group, a methoxy group, a carbonyl carbon, six methine carbons (of which three are oxygenated), seven methylene carbons (one of which is oxygenated), three quaternary carbons, and a methyl carbon. The proton and protonated carbon resonances in the NMR spectra of 1 were assigned by interpretation of 1H NMR, 13C NMR, and HSQC spectroscopic data. In the absence of any other sp or sp2 carbon, the above-mentioned analysis accounted for three out of eight degrees of unsaturation, indicating the structure of 1 must be pentacyclic diterpenoid. The 1H–1H COSY (Fig. 2) correlations of H2-1/H2-2/H2-3, H-5/H2-6/H-7, and H-9/H2-11/H2-12/H-13/H-14 were observed, together with HMBC correlations from H-5 to C-4, C-6, C-9, C-18, C-19, and C-20, from H-9 to C-8, C-11, C-14, C-15, and C-20, and from H-13 to C-8, C-11, C-12, C-14, C-15, and C-16 suggested that 1 is an ent-kaurane diterpenoid. Further analysis of the 2D NMR spectroscopic data of 1 revealed that 1 is a 7,20-epoxyent-kaurane diterpenoid with an acetal moiety at C-20. This assumption was confirmed by the HMBC correlations of H-1, H-5, H-7, and H-9 with C-20 (δC 102.2, d). The acetoxy group was located at C-14 as demonstrated by the HMBC correlations from H-14 (δH 6.19, s) to C-8, C-9, C-12, C-15, C-16, and acetyl carbonyl. C-18 was substituted with hydroxy group as suggested by the HMBC correlations of H2-18 (δH 3.52 and 3.34, 2H, each d, J = 10.4 Hz) with C-3, C-4, C-5, and C-19. HMBC correlations of H-20 (δH 5.30, s) with C-1, C-5, C-7, C-9, and the methoxy carbon indicated that the methoxy group was located at C-20. The relative configuration of 1 was determined via its ROESY spectrum (Fig. 2): the ROESY correlations of H2-18/H-5β
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H.-Y. Wu et al. / Fitoterapia 98 (2014) 192–198
Fig. 1. Chemical structures of compounds 1–7.
and of H-5β/H-9β indicated the β-orientation of H-5, H-9, and C-18, while the ROESY correlations of H-14/H-11α suggested the α-orientation of H-14. Moreover, the ROESY correlation of H-20/Me-19α revealed that the relative configuration of C-20 was S*, and this assignment was also supported by referring to isorosthins C and D [16]. The C-20R* of isorosthin C and C-20S* of isorosthin D were determined by the ROESY correlations of MeO-20/Me-19α and of H-20/Me-19α, respectively, which was confirmed by the single-crystal X-ray diffraction analysis of isorosthin C [16]. For the purpose of determining the absolute configuration of 1, we thus resorted to an X-ray diffraction experiment using the anomalous scattering of Cu Kα radiation yielded a Flack parameter of 0.1(2) [11] and a Hooft parameter of 0.05(11) for
1
H-1H COSY: H
H HMBC : H
3214 Bijvoet pairs, confirming the absolute configuration of 1 as 4S, 5R, 7R, 8R, 9R, 10S, 13S, 14R, and 20S (Fig. 3). Therefore, the structure of 1 was assigned as 20(S)-18-hydroxy-20methoxy-14β-acetoxy-7,20-epoxy-ent-kaur-16-en-15-one. The HREIMS ([M]+ 390.2049, calcd 390.2042) for isowikstroemin B (2) indicated a molecular formula of C22H30O6. The NMR data of 2 closely resemble those of compound 1, but for the downfield shift for H-20 from δH 5.30 in 1 to δH 6.08 in 2 and upfield shift for C-20 from δC 102.2 in 1 to δC 94.5 in 2, together with the absence of the methoxy group suggested that a hydroxy group instead of a methoxy group at C-20 in 2 [12]. This assumption was confirmed by the HMBC correlations of H-20 with C-1, C-5, C-7, C-9, and C-10. The ROESY spectrum of 2 indicated that the relative configurations
C ROESY : H
Fig. 2. 1H–1H COSY, selected HMBC, and key ROESY correlations of 1.
H
H.-Y. Wu et al. / Fitoterapia 98 (2014) 192–198
Fig. 3. The ORTEP drawing of compound 1.
of the stereogenic centers in 2 were identical to those of 1. Compound 2 was thus identified as 20(S*)-18,20-dihydroxy14β-acetoxy-7,20-epoxy-ent-kaur-16-en-15-one. The molecular formula of isowikstroemin C (3) was determined to be C21H30O5 by HREIMS ([M]+ 362.2076, calcd 362.2093), indicating seven degrees of unsaturation. Analysis of its 1D and 2D NMR data suggested that 3 is a 7,20-epoxy-entkauranoid with a methyl group, an olefinic group, a methoxy group, seven methylene (one oxygenated), six methine (three oxygenated), and four quaternary carbons (one carbonyl). The presence of a methoxy group at C-20 and two hydroxy groups at C-14 and C-18 were supported by the HMBC correlations of the protons of methoxy group (δH 3.41, s) with C-20, of H-14 (δH 5.14, s) with C-9, C-12, C-15, and C-16, and of H2-18 (δH 3.52 and 3.36, 2H, each d, J = 10.5 Hz) with C-3, C-4, C-5, and C-19. The relative configuration of 3 was determined by ROESY spectral data analysis. The correlation between H-14 and H-11α suggested the α-orientation of H-14, while the correlation between H2-18 and H-5β demonstrated the β-orientation of the C-18 hydroxymethyl group. Similarly, the correlation between H-20 and Me-19α indicated the relative configuration of C-20 as S*. Therefore, the structure of compound 3 was defined as 20(S*)-14β,18-dihydroxy-20-methoxy-7,20-epoxyent-kaur-16-en-15-one. Isowikstroemin D (4), was isolated as a white amorphous powder, and its molecular formula was established to be C20H28O5 by HREIMS ([M]+ 348.1938, calcd 348.1937). Comparisons of the 1H and 13C NMR data of 4 with those of 3 (Tables 1 and 2) indicated that both compounds have identical skeletons and substitution patterns, differing only in that 4 has a hydroxy group at C-20 rather than a methoxy group in 3, causing downfield shift for H-20 from δH 5.35 in 3 to δH 6.15 in 4 and upfield shift for C-20 from δC 102.5 in 3 to δC 94.6 in 4. This conclusion was verified by the HMBC correlations of H-20 with C-1, C-5, C-7, C-9, and C-10. The ROESY spectrum of 4 indicated that the relative configurations of its stereogenic carbons were identical to those of 3. Compound 4 was thus identified as 20(S*)-14β,18,20-trihydroxy-7,20-epoxy-ent-kaur16-en-15-one.
197
Isowikstroemin E (5) has a molecular formula of C23H34O6 based on HREIMS ([M]+ 406.2353, calcd 406.2355), indicating seven degrees of unsaturation. Except for one acetoxy group, 21 carbon atoms found in the 13C NMR and DEPT spectra consisted of an olefinic quaternary carbon, an olefinic methylene carbon, a methoxy carbon, seven methylene carbons (one oxygenated), seven methine carbons (four oxygenated), three quaternary carbons, and a methyl carbon, which were again consistent with a skeleton of a 7,20-epoxy-ent-kaurane diterpenoid. Two hydroxy groups at C-15 and C-18 based on the HMBC correlations of H-15 (δH 4.78, s) with C-7, C-9, and C-16, and of H2-18 (δH 3.51 and 3.37, 2H, each d, J = 10.5 Hz) with C-3, C-4, C-5, and C-19. A methoxy group and an acetoxy group located at C-20 and C-14, respectively, according to the HMBC correlations from H-20 (δH 5.37, s) to C-1, C-7, C-9, C-10, and methoxy carbon, and from H-14 (δH 5.95, s) to C-8, C-9, C-12, C-16, and acetyl carbonyl. A ROESY experiment revealed that H-14 and H-15 in 5 were both α-oriented based on the correlations of H-14/H-11α, and of H-15/H-13α, while C-18 was β-oriented based on the correlation of H2-18/H-5β. The relative configuration of C-20 was assigned as S* according to the ROESY correlation of H-20 and Me-19α. Therefore, the structure of 5 was defined as 20(S*)-15β,18-dihydroxy-20methoxy-14β-acetoxy-7,20-epoxy-ent-kaur-16-ene. The HREIMS ([M]+ 392.2185, calcd 392.2199) for isowikstroemin F (6) indicated a molecular formula of C22H32O6. The 1H and 13C NMR data of 6 were similar to those of 5 (Tables 1 and 2) except that the hydroxy group at C-20 in 6 instead of the methoxy group in 5. Downfield shift for H-20 from δH 5.37 in 5 to δH 6.15 in 6 and upfield shift for C-20 from δC 102.9 in 5 to δC 95.1 in 6, together with the HMBC correlations of H-20 with C-5, C-7, C-9, and C-10 confirmed this assumption. In addition, correlations observed in the ROESY spectrum indicated that the orientations of the substituents in 6 were the same as in 5. The structure of compound 6 was thus defined as 20(S*)-15β, 18, 20-trihydroxy-14βacetoxy-7,20-epoxy-ent-kaur-16-ene. The molecular formula of isowikstroemin G (7) was determined to be C20H30O4 by HREIMS ([M]+ 334.2144, calcd 334.2144), indicating six degrees of unsaturation. Its IR absorption bands at 3392 and 1635 cm−1 indicated the presence of hydroxy and alkenyl groups. Its 13C NMR and DEPT data (Table 2) exhibited 20 carbon resonances including an olefinic group, a methyl group, nine methylene (two of which are oxygenated), four methine (one of which is oxygenated), and four quaternary carbons (which one is oxygenated). Protons were assigned to related carbon resonances via the 1H NMR, 13C NMR, and HSQC spectroscopic data. Based on its NMR data, compound 7 is also a 7,20-epoxy-ent-kaurane diterpenoid. The presence of two hydroxy groups at C-15 and C-18 in 7 was confirmed by the observation of HMBC correlations from H-9, H-13, H-14, and H-17 to C-15 (δC 75.9, d), and from H2-18 (δH 3.52 and 3.40, 2H, each d, J = 10.4 Hz) to C-3, C-4, C-5, and C-19. In addition, HMBC correlations of H2-20 (δH 4.36 and 4.08, 2H, each d, J = 9.3 Hz) with C-5, C-7 (δC 96.7, s), C-9, and C-10 suggested that C-20 connects with C-7 through an oxygen atom and forms the structure of hemiacetal at C-7. The ROESY correlation from H-15 to H-13α indicated that the C-15 hydroxy group in 7 was β-oriented. Therefore, the structure of compound 7 was assigned as 7β,15β,18-trihydroxy-7,20epoxy-ent-kaur-16-ene.
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Table 3 IC50 values (μM) of diterpenoids from I. wikstroemioides against Tumor Cell Lines.a Compound
HL-60
SMMC-7721
A-549
MCF-7
SW-480
1 2 3 4 5 6 7 DDPb Paclitaxelb
2.51 ± 0.16 2.48 ± 0.11 3.79 ± 0.06 2.97 ± 0.14 N40 N10 ± 1.22 N10 ± 0.54 1.75 ± 0.04 b0.008
1.02 ± 0.02 3.78 ± 0.02 4.72 ± 0.09 3.65 ± 0.07 N40 N10 ± 0.16 N10 ± 0.32 4.46 ± 0.08 b0.008
0.88 ± 0.04 3.34 ± 0.04 7.00 ± 0.20 3.84 ± 0.13 N40 N10 ± 1.02 N10 ± 0.75 7.61 ± 0.22 b0.008
2.1 ± 0.10 2.28 ± 0.07 5.35 ± 0.28 3.37 ± 0.06 N40 N40 4.79 ± 0.21 15.7 ± 0.08 b0.008
1.22 ± 0.08 2.40 ± 0.15 4.23 ± 0.11 2.51 ± 0.13 N40 N10 ± 0.48 4.23 ± 0.01 15.0 ± 0.03 b0.008
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. Each value represents the mean ± SEM (n = 3). b DDP (cis-platin) and paclitaxel were used as positive controls.
Table 4 IC50 values (μM) of diterpenoids from I. wikstroemioides against LPS-activated NO production in RAW264.7 cells.a
Appendix A. Supplementary data
Compound
IC50 (μM)
Compound
IC50 (μM)
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.08.012.
1 3 6 MG-132
1.34 1.49 23.6 0.13
2 4 7
2.28 ± 0.17 1.13 ± 0.14 2.50 ± 0.37
References
± ± ± ±
0.11 0.08 0.41 0.07
a Each value represents the mean ± SEM (n = 3). Compounds with values of IC50 b 10 µM exhibited interesting inhibitory activity against NO production.
All of the isolates obtained from I. wikstroemioides in this work were evaluated for their cytotoxicity against the HL-60 (acute leukemia), SMMC-7721 (hepatic cancer), A-549 (lung cancer), MCF-7 (breast cancer), and SW-480 (colon cancer) human tumor cell lines using the MTS method [13], with cisplatin and paclitaxel as positive controls. Compounds 1–4 exhibited significant cytotoxic activity with IC50 values ranging from 0.9 to 7.0 μM, while compound 7 showed selective antiproliferative activity against the MCF-7 and SW-480 cancer cell lines, with IC50 values of 4.8 μM and 4.2 μM, respectively (Table 3). In addition, six isolates were tested for their capacity to inhibit NO production in LPS-stimulated RAW264.7 cells. Interesting inhibitory activity was observed for compounds 1, 2, 3, 4, and 7 with IC50 values ranging from 1.1 to 2.5 μM (Table 4). 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), the Major Direction Projection Foundation of CAS Intellectual Innovation Project (Grant KSCX2-EW-J-24), and the West Light Foundation of the Chinese Academy of Sciences (J.-X. P.).
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