Article pubs.acs.org/jnp
Anti-inflammatory Labdane Diterpenoids from Lagopsis supina Hui Li,†,‡,# Man-Man Li,†,‡,# Xiao-Qin Su,†,‡ Jing Sun,†,‡ Yu-Fan Gu,†,‡ Ke-Wu Zeng,⊥ Qian Zhang,† Yun-Fang Zhao,† Daneel Ferreira,§ Jordan K. Zjawiony,§ Jun Li,*,† and Peng-Fei Tu*,† †
Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China ‡ School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, People’s Republic of China ⊥ State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, People’s Republic of China § Department of Pharmacognosy and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677-1848, United States S Supporting Information *
ABSTRACT: Ten new labdane diterpenoids, lagopsins A−H (1−3, 5, 7−10) and 15-epi-lagopsins C and D (4, 6), together with five known labdane diterpenoids (11−15), were isolated from the whole plants of Lagopsis supina. The absolute configuration of lagopsin A (1) was determined by singlecrystal X-ray crystallographic analysis. Compounds 7, 9, 13, and 15 exhibited moderate inhibition of nitric oxide production stimulated by lipopolysaccharide in BV-2 microglial cells with IC50 values in the range 14.9−34.9 μM.
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isolation and structural elucidation of the new compounds are described as well as their inhibitory effects on NO production in LPS-stimulated BV-2 microglial cells.
abdane diterpenoids are mainly distributed in higher plants such as the Acanthaceae, Asteraceae, Chloranthaceae, Cistaceae, Cupressaceae, Euphorbiaceae, Labiatae, Pinaceae, Solanaceae, Verbenaceae, and Zingiberaceae. Significant biological properties, such as anti-inflammatory, antibacterial, antifungal, antiprotozoal, cytotoxic, cardiotonic, antiviral, enzyme inducing, and hypotensive activities and modulation of immune cell functions, have been ascribed to the labdanes.1,2 Lagopsis supina (Stephan.) Ik.-Gal., family Lamiaceae (syn. Labiatae), is a perennial herbaceous plant widely distributed in northeast Asia. The whole plant, also known as “Xiazhicao” in Chinese, has been used in traditional Chinese medicine for the treatment of gynecologic disorders such as menorrhagia, irregular menstruation, and painful menstruation, as well as edema in acute and chronic nephritis.3,4 A wide array of pharmacological activities including improvement of blood and lymph microcirculation,5−7 myocardioprotective,8 anti-inflammatory,9 and antioxidative10 effects have been reported for the crude extracts of this plant. Previous phytochemical investigations have resulted in the isolation of flavonoid glycosides,11 phenylethanoid glycosides,12 and phytosterols.13 As part of an ongoing effort to search for bioactive diterpenoids from medicinal plants, the acetone extract of the whole plants of L. supina was investigated. This procedure led to the isolation of 10 new labdane diterpenoids, named lagopsins A−H (1−3, 5, 7−10) and 15-epi-lagopsins C and D (4, 6), together with five known labdane diterpenoids, namely, 7β-acetoxy-15,16-epoxylabda-13(16),14-diene-6β,9α-diol (11),14 15,16-epoxylabda13(16),14-diene-6β,7β,9α-triol (12),14 leoleorin A (13),15 leoheterin (14),14,16 and leoleorin B (15).15 Herein, the © 2014 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION The acetone extract of the whole plants of L. supina was dissolved in 90% aqueous MeOH and partitioned with hexanes and CH2Cl2, successively. The CH2Cl2-soluble portion was repeatedly subjected to silica gel, Sephadex LH-20, and Lichroprep RP-C18 gel column chromatography and semipreparative RP-HPLC, to afford 10 new (1−10) and five known (11−15) labdane diterpenoids. Lagopsin A (1) was obtained as colorless plates via crystallization from MeOH, [α]21D +20. The positive-ion HRESIMS gave a pseudomolecular ion at m/z 401.2314 [M + Na]+ that together with 13C NMR spectroscopic data was consistent with a molecular formula of C22H34O5, indicating six indices of hydrogen deficiency. The IR spectrum showed absorption bands for hydroxy (3450 cm−1) and ester (1720 cm−1) functionalities. The 1H NMR data (Table 1) displayed singlets for three tertiary methyl groups at δH 1.00 (6H, s, CH3 × 2), and 1.27 (3H, s), one secondary methyl group at δH 1.12 (3H, d, J = 6.5 Hz), and resonances of three olefinic protons at δH 7.35 (1H, br s), 7.23 (1H, br s), and 6.27 (1H, br s). The 13 C NMR data of 1 showed 22 carbon resonances comprising an ester carbonyl (δC 172.4), a quaternary sp2 (δC 125.2), three quaternary sp3 (δC 78.3, 43.5, 33.9), three sp2 methine (δC Received: February 11, 2014 Published: April 8, 2014 1047
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142.9, 138.5, 110.7), four sp3 methine (δC 72.8, 72.4, 46.0, 38.3), five sp3 methylene (δC 43.7, 35.1, 34.1, 21.5, 18.6), and five methyl (δC 33.6, 23.8, 21.7, 19.5, 11.0) carbons. The 1D and 2D NMR spectroscopic data revealed that compound 1 belongs to the labdane class of diterpenoids possessing a furan moiety.15,17,18 The 1H and 13C NMR spectroscopic data of 1 were comparable to those of the semisynthetic 15,16epoxylabda-13(16),14-diene-6β,7β,9α-triol (12),14 except for the presence of resonances of an acetoxy group (δH 2.10, δC 172.4, 21.7) in 1. The deshielded H-6 resonance (δH 5.59, br s) indicated that the acetoxy group is located at C-6, as confirmed by the HMBC correlation between H-6 and the acetoxy carbonyl carbon. The NOESY data revealed that the relative configurations at C-5, -6, -7, -8, -9, and -10 of 1 are the same as those of 15,16-epoxylabda-13(16),14-diene-6β,7β,9α-triol (12). The absolute configuration of 1 was defined as (5S,6S,7R,8R,9R,10S) by single-crystal X-ray diffraction analysis (Figure 1). Therefore, the structure of 1 was established as (5S,6S,7R,8R,9R,10S)-6β-acetoxy-15,16-epoxylabda-13(16),14diene-7β,9α-diol, and this compound has been named lagopsin A. Lagopsin B (2) was obtained as a colorless gum, and its molecular formula was determined as C24H36O6 by the 13C NMR spectroscopic data and the HRESIMS showing a pseudomolecular ion at m/z 443.2416 [M + Na]+. The 1D Table 1. 1H NMR Data of Compounds 1−10 (δ in ppm, J in Hz, 500 MHz, in CDCl3) position
1a
1a 1b 2a 2b 3a 3b 5
1.47, m 1.47, m 1.62, m 1.51, m 1.35, m 1.19, m 1.75, d (1.5)
1.47, 1.47, 1.62, 1.51, 1.36, 1.21, 1.80,
6 7
5.59, br s 3.65, dd (11.0, 3.5) 2.07, m 1.86, m 1.84, m 2.56, m 2.50, m 6.27, br s
5.67, br s 4.90, dd (11.0, 3.5) 2.29, m 1.91, m 1.85, m 2.58, m 2.50, m 6.29, br s
8 11a 11b 12a 12b 14a
2a m m m m m m br s
14b 15
7.35, br s
7.37, br s
16a
7.23, br s
7.24, br s
17
1.12, d (6.5)
0.99, d (6.5)
18 19 20 22 23 24
1.00, 1.00, 1.27, 2.10,
1.00, 0.98, 1.29, 2.08,
16b
a
s s s s
s s s s
3/4a 1.36, m 1.28, m 1.62, m 1.53, m 1.35, m 1.19, m 1.57, d (1.5)/1.53, d (1.8) 5.60, t (3.0) 3.62,dd (11.0, 3.5)/ 3.58, dd (11.0, 3.5) 2.09, m 2.12, m 1.80, m 2.25, m/2.10, m 2.25, m/2.00, m 2.48, dd (13.5, 5.5)/ 2.16, dd (13.5, 5.5) 2.02, m/2.30, dd (13.5, 1.5) 5.56, d (5.0)/5.43, d (3.5) 3.98, d (8.5)/4.18, d (8.5) 3.79, d (8.5)/3.53, d (8.5) 1.04, d (7.0)/1.06, d (7.0) 0.99, s 0.98, s/0.99, s 1.22, s/1.24, s 2.11, s
5/6a 1.47, 1.42, 1.66, 1.52, 1.36, 1.17, 1.58,
m m m m m m br s/1.41, br s
5.61, br s 3.62, dd (11.0, 3.5)/ 3.67, dd (11.0, 3.5) 2.11, m 2.20, m 1.80, m 2.21, m/2.19, m 2.21, m/1.90, m 2.36, m/2.42, m
7a 1.47, 1.42, 1.65, 1.52, 1.36, 1.20, 1.77,
m m m m m m br s
8a 1.45, 1.37, 1.60, 1.50, 1.47, 1.32, 2.77,
m m m m m m d (3.0)
9a 1.50, 1.37, 1.63, 1.52, 1.46, 1.32, 2.73,
m m m m m m d (3.0)
10a 1.98, 1.66, 1.77, 1.65, 1.55, 1.40,
m m m m m m
5.61, br s 3.66, dd (11.0, 3.0) 2.06, m 1.82, t (8.0) 1.82, t (8.0) 2.44, m 2.44, m 6.79, br s
5.39, d (3.0)
5.38, d (2.5)
6.19, s
2.55, 2.75, 2.75, 2.60, 2.60, 6.27,
2.58, 2.84, 2.84, 2.53, 2.47, 7.19,
2.27, 1.92, 2.74, 2.67, 6.32,
5.75, br s
7.32, br s
m, 2.46, m m m m m br s
m, 2.44, m m m m m br s
m m m m br s
1.93, m/1.91, m 5.61, br s/5.42, d (4.0) 4.05, d (8.5)/4.27, d (9.0) 4.00, d (8.5)/3.74, d (9.0) 1.01, d (6.0)/1.00, d (6.0) 0.99, s 0.98, s 1.23, s/1.26, s 2.11, s
4.77, br s (2H)
7.22, br s
7.36, br s 7.27, br s
1.11, d (6.5)
1.05, t (7.5)
1.05, t (7.5)
1.44, s
1.00, 1.00, 1.25, 2.12, 3.59,
1.00, 1.05, 1.37, 2.06,
1.03, 1.06, 1.43, 2.09,
1.24, s 1.21, s 1.33, s
s s s s s
s s s s
s s s s
1.99, s
Assignments were based on HSQC and HMBC experiments. 1048
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confirmed by the HMBC correlation between H-7 (δH 4.90, dd, J = 11.0, 3.5 Hz) and the acetoxy carbonyl carbon (δC 170.5). Thus, the structure of 2 (lagopsin B) was identified as 6β,7βdiacetoxy-15,16-epoxylabda-13(16),14-diene-9α-ol. Lagopsin C (3) and 15-epi-lagopsin C (4) were obtained as an inseparable mixture (1:2), [α]21D −17. The positive-ion HRESIMS data showed a pseudomolecular ion at m/z 419.2402 [M + Na]+ and, in conjunction with the 13C NMR data, established a molecular formula of C22H36O6, implying five indices of hydrogen deficiency. The IR spectrum of 3/4 indicated the presence of hydroxy (3440 cm−1) and ester carbonyl (1736 cm−1) functional groups. The 1H and 13C NMR data (Tables 1 and 2) showed duplicate resonances for three tertiary methyl groups (δH 0.98/0.99, 0.99 × 2, 1.22/1.24; δC 19.8/19.9, 33.0/33.1, 23.9 × 2, respectively), one secondary methyl group (δH 1.04/1.06; δC 11.6/11.8), two oxygenated quaternary carbons (δC 93.2/93.7, 89.2/90.3), a hemiacetal methine (δH 5.56/5.43; δC 98.3/98.5), an isolated oxygenated methylene (δH 3.98/4.18, 3.79/3.53; δC 76.8/75.8), two oxygenated methines (δH 5.60 × 2, 3.62/3.58; δC 72.7/72.5, 73.2/73.1, respectively), six methylenes (δC 47.3/46.5, 44.0/ 43.9, 38.3/35.9, 34.1/34.3, 30.0/29.7, 18.6 × 2), two methines (δC 47.4/47.5, 38.4/38.2), and two quaternary carbons (δC 42.8 × 2, 34.0/34.2). These data suggested that compounds 3/4 are bis-spirolabdane diterpenoids possessing a 1,6-dioxaspiro[4.4]nonane C/D ring moiety.17,19−21 In addition, duplicate resonances of an acetoxy group (δH 2.11 × 2; δC 172.5/ 172.4, 21.7 × 2) were evident in the NMR spectra. Comparison of NMR data of 3/4 with those of lagopsin A (1) suggested that they share the same A- and B-ring substitution patterns. In turn, comparison of the 13C NMR chemical shifts with those of bis-spirolabdane diterpenoids15,19−21 supported the 1,6dioxaspiro[4.4]nonane moiety of 3/4. The molecular structures
Figure 1. ORTEP drawing of compound 1 (ORTEP diagram is one of two independent molecules of compound 1).
NMR data (Tables 1 and 2) of 2 were similar to those of 1, except for the presence of an acetoxy rather than a hydroxy group in 2. The C-7 location of the acetoxy group was
Table 2. 13C NMR Data of Compounds 1−11 (δ in ppm, 125 MHz, in CDCl3) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 a
1a 34.1, 18.6, 43.7, 33.9, 46.0, 72.4, 72.8, 38.3, 78.3, 43.5, 35.1, 21.5, 125.2, 110.7, 142.9, 138.5, 11.0, 33.6, 23.8, 19.5, 172.4, 21.7,
CH2 CH2 CH2 C CH CH CH CH C C CH2 CH2 C CH CH CH CH3 CH3 CH3 CH3 C CH3
2a 34.0, 18.6, 43.6, 34.0, 46.0, 69.2, 74.3, 36.3, 78.3, 43.5, 35.0, 21.5, 125.0, 110.7, 143.1, 138.5, 10.8, 33.5, 23.7, 19.4, 170.5, 21.5, 170.5, 20.9,
CH2 CH2 CH2 C CH CH CH CH C C CH2 CH2 C CH CH CH CH3 CH3 CH3 CH3 C CH3 C CH3
3/4a
5/6a
7a
34.1/34.3, CH2 18.6/18.6, CH2 44.0/43.9, CH2 34.0/34.2, C 47.4/47.5, CH 72.7/72.5, CH 73.2/73.1, CH 38.4/38.2, CH 93.2/93.5, C 42.8/42.8, C 30.0/29.7, CH2 38.3/35.9, CH2 89.5/90.3, C 47.3/46.5, CH2 98.3/98.5, CH 77.8/76.3, CH2 11.6/11.8, CH3 33.0/33.1, CH3 23.9/23.9, CH3 19.8/19.9, CH3 172.5/172.4, C 21.7/21.7, CH3
34.2/34.4, CH2 18.7/18.7, CH2 44.1/44.2, CH2 34.1/34.4, C 47.3/48.1, CH 72.9/72.4, CH 73.1/72.8, CH 38.3/38.3, CH 93.3/93.5, C 42.8/42.8, C 30.0/29.8, CH2 38.7/35.0, CH2 89.1/90.6, C 47.9/45.4, CH2 99.3/99.6, CH 78.4/77.4, CH2 11.6/11.8, CH3 33.0/33.2, CH3 23.8/23.8, CH3 19.8/19.9, CH3 172.3/172.3, C 21.7/21.7, CH3
34.1, 18.6, 43.6, 34.0, 46.1, 72.3, 72.9, 38.5, 77.9, 43.7, 32.3, 21.9, 139.0, 141.7, 102.7, 171.6, 10.9, 33.6, 23.8, 19.4, 172.4, 21.7, 57.1,
CH2 CH2 CH2 C CH CH CH CH C C CH2 CH2 C CH2 CH C CH3 CH3 CH3 CH3 C CH3 CH3
8a 39.3, 18.2, 41.8, 34.2, 47.7, 77.4, 208.3, 32.7, 215.0, 51.4, 37.7, 19.0, 124.3, 111.1, 142.6, 139.0, 7.4, 33.5, 24.6, 19.9, 170.0, 20.9,
CH2 CH2 CH2 C CH CH C CH2 C C CH2 CH2 C CH CH CH CH3 CH3 CH3 CH3 C CH3
9a 39.8, 18.2, 42.0, 34.6, 47.7, 77.3, 208.3, 32.7, 214.7, 51.5, 34.3, 19.9, 133.1, 145.5, 70.1, 174.2, 7.4, 33.6, 24.5, 19.9, 169.9, 20.9,
CH2 CH2 CH2 C CH CH C CH2 C C CH2 CH2 C CH CH2 C CH3 CH3 CH3 CH3 C CH3
10a 31.8, 17.1, 38.5, 37.1, 178.6, 121.4, 201.8, 78.3, 78.0, 47.3, 30.5, 20.8, 125.6, 111.0, 142.8, 138.6, 24.4, 32.0, 31.0, 27.0,
CH2 CH2 CH2 C C CH C C C C CH2 CH2 C CH CH CH CH3 CH3 CH3 CH3
11a 34.1, 18.7, 43.4, 34.3, 47.3, 68.7, 77.6, 35.8, 78.5, 43.3, 34.9, 21.4, 125.2, 110.8, 143.0, 138.5, 11.0, 33.7, 24.6, 19.6, 170.2, 21.2,
CH2 CH2 CH2 C CH CH CH CH C C CH2 CH2 C CH CH CH CH3 CH3 CH3 CH3 C CH3
Assignments were based on HSQC and HMBC experiments. 1049
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its 13C NMR and HRESIMS data (m/z 447.2359 [M + Na]+). The IR spectrum showed absorptions characteristic of hydroxy (3439 cm−1) and ester carbonyl (1766 and 1728 cm−1) groups. The 1H NMR data (Table 1) displayed singlets for three tertiary methyl groups at δH 1.00 (6H, s, CH3 × 2) and 1.25 (3H, s) and one secondary methyl group at δH 1.11 (3H, d, J = 6.5 Hz), indicative of 7 being a labdane-type diterpenoid.15,22,23 The 1H and 13C NMR data (Tables 1 and 2) were comparable to those of 1, with similar resonances for the A and B rings. The 13 C NMR resonances at δC 171.6, 141.7, 139.0, 102.7, and 57.1 and the 1H NMR resonances at δH 6.79 (1H, br s), 5.75 (1H, br s), and 3.59 (3H, s) indicated that 7 possesses a 3substituted 5-methoxyfuran-2(5H)-one moiety at C-12 rather than the furan ring of 1. A comparison of the NMR spectroscopic data of the C-11−C-16 region to those reported for the structural analogues sibiricinone C22 and 6β-acetoxy-9αhydroxy-15-methoxy-13(14)-labden-16,15-olide24 facilitated assembly of its C-11−C-16 component. The molecular structure of 7 was confirmed by the HMBC correlations between H3-20 and C-1/C-5/C-9; H3-18 and C-4/C-5/C-19; H3-17 and C-7/ C-8/C-9; H-15 and C-13/C-16/C-23; H-14 and C-13/C-15/ C-16; H2-12 and C-13/C-14; H2-11 and C-8/C-9/C-10/C-12/ C-13; H-8 and C-6/C-7/C-17; H-6 and C-5/C-7/C-8/C-10; and H3-23 and C-15 (Figure 3). The relative configurations at
of 3/4 were confirmed by the HMBC correlations between H320 and C-1/C-5/C-9; H3-18 and C-4/C-5/C-19; H3-17 and C7/C-8/C-9; H2-16 and C-12/C-15; H-15 and C-13/C-14/C16; H2-14 and C-12/C-13/C-16; H2-11 and C-8/C-10/C-13; and H-6 and C-5/C-7/C-8/C-10 (Figure 2). The location of
Figure 2. Selected HMBC (arrows point from protons to carbons) and NOE correlations of compounds 3/4.
the acetoxy group at C-6 was confirmed by the HMBC correlation between H-6 (δH 5.60, br s) and the acetoxy carbonyl carbon (δC 172.5/172.4). In the NOESY spectrum, the NOEs from H3-20 to H3-19 and from H-5 to H3-18 supported the trans A/B junction in 3/4. NOEs between H-5/ H-6, H-5/H-1α, H-6/H-7, and H-7/H3-17 indicated that these hydrogens are cofacial and α-oriented, while correlations between H3-20/H-8, H2-11, and H-8/H2-11 suggested that these protons are β-cofacially oriented. The NOE correlations between H2-14/H3-17 and H-1α/H2-16 suggested the relative C-13 configuration as shown in 3/4 (Figure 2). This observation also proved the α-orientation of Me-17. Furthermore, the NOE correlation between H-15 and H2-12 suggested the α-orientation of the HO-15 in 4, while the absence of a similar correlation indicated a β-oriented 15-OH group in 3. Accordingly, the structures of lagopsin C (3) and 15-epi-lagopsin C (4) were established as shown. An inseparable 7:10 epimeric mixture of lagopsin D (5) and 15-epi-lagopsin D (6) was obtained as a colorless gum, [α]21D −7. The IR spectrum exhibited absorption bands for hydroxy (3440 cm−1) and ester carbonyl (1735 cm−1) groups. The molecular formula C22H36O6 of 5/6 was determined to be the same as that of 3/4 from the 13C NMR and positive-ion HRESIMS data (m/z 419.2414 [M + Na]+). Slight differences in the 1H and 13C NMR spectroscopic values (Tables 1 and 2) of 5/6 in comparison with those of 3/4 suggested that they could be stereo- or regioisomeric compounds. 2D NMR experiments facilitated the assembly of the molecular structures of 5/6 as the same as those of 3/4. The relative configurations at C-5, -6, -7, -8, -9, and -10 were the same as assigned for 3/4 on the basis of NOESY data. The NOE correlations between H2-16/H3-17 and H-1α/H2-14 in compounds 5/6 in contrast to H2-14/H3-17 and H-1α/H2-16 in 3/4 indicated their regioisomeric nature. The respective α- and β-orientations of the 15-OH in 6 and 5 were assigned via the NOE correlation between H-15 and H2-12 in 6 and the absence of a similar association in 5. Therefore, the structures of lagopsin D (5) and 15-epi-lagopsin D (6) were defined as shown in 5/6. In view of the exposure of hemiacetals 3/4 and 5/6 to methanol, it is surprising that the corresponding acetals were absent. Lagopsin E (7) was isolated as a colorless gum, [α]21D +13, and its molecular formula was determined to be C23H36O7 via
Figure 3. Selected HMBC (arrows point from protons to carbons) correlations of compounds 7 and 8.
C-5, -6, -7, -8, -9, and -10 as assigned via NOE association were the same as determined for 1. However, the C-15 relative configuration could not be assigned. Thus, the structure of 7 was defined as 6β-acetoxy-7β,9α-dihydroxy-15-methoxy13(14)-labden-16,15-olide, named lagopsin E. Lagopsin F (8) was obtained as a colorless, viscous oil, [α]21D +9. Its 13C NMR and positive-ion HRESIMS data, exhibiting a sodiated molecular ion at m/z 399.2143, corresponded to a molecular formula of C22H32O5 (calcd for C22H32O5Na, 399.2142), with seven indices of hydrogen deficiency. The IR spectrum showed absorption bands at 1743, 1729, and 1698 cm−1, reminiscent of the presence of the ester and keto carbonyl functionalities. The 1H and 13C NMR spectra exhibited resonances for three tertiary methyl groups (δH 1.00, 1.05, 1.37; δC 33.5, 24.6, 19.9, respectively), a propanoyl group [δH 1.05 (3H, t, J = 7.5 Hz), 2.55, 2.46 (each 1H, m); δC 7.4, 32.7, 208.3], an acetoxy group (δH 2.06; δC 170.0, 20.9), a carbonyl group (δC 215.0), a β-substituted furan (δH 7.32, 7.22, 6.27; δC 111.1, 124.3, 139.0, 142.6), five methylenes, two methines (one oxygenated), and two quaternary carbons. These spectroscopic data indicated 8 to be an 8,9-seco-labdane diterpenoid with an acetoxy substituent.25,26 The 1D NMR data of 8 resembled those of the dioxo derivative of hispanolone26 except for the resonances of an acetoxy group in 8. The acetoxy group was assigned to C-6 via the HMBC correlation between H-6 and the acetoxy carbonyl carbon. The proposed structure of 8 was further confirmed by the HMBC 1050
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By comparing spectroscopic and specific rotation data with literature values, the remaining three known compounds were identified as leoleorin A (13),15 leoheterin (14),14,16 and leoleorin B (15).15 Compounds 1−15 were evaluated for their inhibitory effects on the NO production in LPS-stimulated BV-2 microglial cells using the Griess assay.27,28 Compounds 7, 9, 13, and 15 showed moderate inhibitory activity against NO production with IC50 values of 16.2, 34.9, 14.9, and 34.0 μM, respectively. Quercetin was used as a positive control (IC50 = 10.0 μM) (Table 3). Compounds 1−6, 8, 10−12, and 14 were inactive
correlations between H3-20 and C-1/C-5/C-9/C-10; H3-18 and C-19/C-4/C-5; H3-17 and C-7/C-8; H-16 and C-13/C14/C-15; H-15 and C-13/C-14/C-16; H-14 and C-13/C-15/ C-16; H2-12 and C-9/C-11/C-13/C-14/C-16; H2-11 and C-9/ C-12/C-13; H-8 and C-7/C-17; and H-6 and C-4/C-5/C-7/C10 (Figure 3). The NOE correlation between H-6 and H3-20 indicated that CH3-20 and the C-6 side chain are cofacial. However, the C-6 relative configuration could not be assigned. Therefore, the structure of lagopsin F (8) was identified as shown. This compound may be derived from the 7-oxo derivative of compound 1 via a retro-aldol reaction with concomitant cleavage of the C-8−C-9 bond. Lagopsin G (9) was obtained as a colorless, viscous oil, [α]21D +15. Its molecular formula was established as C22H32O6 based on the 13C NMR data and a protonated molecular ion [M + H]+ at m/z 393.2256 (calcd for C22H33O6, 393.2272) in its HRESIMS. The IR spectrum exhibited absorption bands for ester (1744 cm−1) and keto carbonyl (1730, 1698 cm−1) functional groups. Comparison of their NMR data suggested that compound 9 differed from compound 8 by replacement of the β-substituted furan ring by a 3-substituted furan-2(5H)-one moiety [δH 4.77 (2H, br s), 7.19; δC 70.1, 133.1, 145.5, 174.2)],23 which was supported by the HMBC correlations between H-15 and C-13/C-14; H-14 and C-13; and H-12 and C-9/C-11/C-13/C-14/C-16. The relative configurations of C-5 and C-10 were the same as those of 8 based on the NOESY data, but the C-6 relative configuration could not be assigned. Accordingly, the structure of lagopsin G (9) was established as shown. Lagopsin H (10) was obtained as a white, amorphous powder, [α]25D −67, and its 13C NMR and positive-ion HRESIMS data showing a pseudomolecular ion at m/z 355.1879 [M + Na]+ established a molecular formula of C20H28O4, indicating seven indices of hydrogen deficiency. The IR spectrum indicated the presence of hydroxy (3431 cm−1) and α,β-unsaturated carbonyl (1660 cm−1) functionalities. The 1 H NMR data (Table 1) displayed singlets for four tertiary methyl groups at δH 1.44, 1.33, 1.24, and 1.21 and the resonances of four olefinic protons at δH 7.36 (1H, br s), 7.27 (1H, br s), 6.32 (1H, br s), and 6.19 (1H, s). The 13C NMR spectrum showed 20 carbon resonances including four methyls, five methylenes, four olefinic methines, and seven quaternary carbons (one carbonyl, two olefinic, and two oxygenated). These spectroscopic data revealed that compound 10 is a labdane diterpenoid with a furan moiety and an α,β-unsaturated carbonyl group.15 The 1D NMR data of 10 (Table 1) were nearly superimposable on those of leoleorin A (13), previously isolated from Leonotis leonurus,15 except for the significantly deshielded C-8 (δC 78.3; ΔδC +30.4) and C-17 (δC 24.4; ΔδC +14.5) resonances. This is in agreement with the addition of a C-8 hydroxy group in 10, which was supported by the presence of a methyl singlet in 10 instead of a doublet for H3-17 in 13. Thus, the structure of 10 (lagopsin H) was defined as 15,16epoxy-8β,9α-dihydroxylabda-5,13(16),14-trien-7-one. Compounds 11 and 12 were earlier reported as intermediates 7β-acetoxy-15,16-epoxylabda-13(16),14-diene-6β,9αdiol (11) and 15,16-epoxylabda-13(16),14-diene-6β,7β,9α-triol (12) in the syntheses of sibiricinone A and leoheterin.14 This is the first report of their natural occurrence. Owing to their structural similarity to compounds 1−10, the trivial names lagopsins I and J were assigned to 11 and 12. The specific rotation and 13C NMR data of lagopsin I (11) are presented herein for the first time.
Table 3. Inhibitory Effects of Compounds from L. supina on LPS-Activated NO Production in BV-2 Microglial Cells compounda 7 9 13 15 quercetinc
IC50 (μM)b 16.2 34.9 14.9 34.0 10.0
± ± ± ± ±
3.2 4.5 2.1 3.8 2.0
a
Compunds 1−6, 8, 10−12, and 14 were inactive (
Anti-inflammatory Labdane Diterpenoids from Lagopsis supina Hui Li,†,‡,# Man-Man Li,†,‡,# Xiao-Qin Su,†,‡ Jing Sun,†,‡ Yu-Fan Gu,†,‡ Ke-Wu Zeng,⊥ Qian Zhang,† Yun-Fang Zhao,† Daneel Ferreira,§ Jordan K. Zjawiony,§ Jun Li,*,† and Peng-Fei Tu*,† †
Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China ‡ School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, People’s Republic of China ⊥ State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, People’s Republic of China § Department of Pharmacognosy and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677-1848, United States S Supporting Information *
ABSTRACT: Ten new labdane diterpenoids, lagopsins A−H (1−3, 5, 7−10) and 15-epi-lagopsins C and D (4, 6), together with five known labdane diterpenoids (11−15), were isolated from the whole plants of Lagopsis supina. The absolute configuration of lagopsin A (1) was determined by singlecrystal X-ray crystallographic analysis. Compounds 7, 9, 13, and 15 exhibited moderate inhibition of nitric oxide production stimulated by lipopolysaccharide in BV-2 microglial cells with IC50 values in the range 14.9−34.9 μM.
L
isolation and structural elucidation of the new compounds are described as well as their inhibitory effects on NO production in LPS-stimulated BV-2 microglial cells.
abdane diterpenoids are mainly distributed in higher plants such as the Acanthaceae, Asteraceae, Chloranthaceae, Cistaceae, Cupressaceae, Euphorbiaceae, Labiatae, Pinaceae, Solanaceae, Verbenaceae, and Zingiberaceae. Significant biological properties, such as anti-inflammatory, antibacterial, antifungal, antiprotozoal, cytotoxic, cardiotonic, antiviral, enzyme inducing, and hypotensive activities and modulation of immune cell functions, have been ascribed to the labdanes.1,2 Lagopsis supina (Stephan.) Ik.-Gal., family Lamiaceae (syn. Labiatae), is a perennial herbaceous plant widely distributed in northeast Asia. The whole plant, also known as “Xiazhicao” in Chinese, has been used in traditional Chinese medicine for the treatment of gynecologic disorders such as menorrhagia, irregular menstruation, and painful menstruation, as well as edema in acute and chronic nephritis.3,4 A wide array of pharmacological activities including improvement of blood and lymph microcirculation,5−7 myocardioprotective,8 anti-inflammatory,9 and antioxidative10 effects have been reported for the crude extracts of this plant. Previous phytochemical investigations have resulted in the isolation of flavonoid glycosides,11 phenylethanoid glycosides,12 and phytosterols.13 As part of an ongoing effort to search for bioactive diterpenoids from medicinal plants, the acetone extract of the whole plants of L. supina was investigated. This procedure led to the isolation of 10 new labdane diterpenoids, named lagopsins A−H (1−3, 5, 7−10) and 15-epi-lagopsins C and D (4, 6), together with five known labdane diterpenoids, namely, 7β-acetoxy-15,16-epoxylabda-13(16),14-diene-6β,9α-diol (11),14 15,16-epoxylabda13(16),14-diene-6β,7β,9α-triol (12),14 leoleorin A (13),15 leoheterin (14),14,16 and leoleorin B (15).15 Herein, the © 2014 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION The acetone extract of the whole plants of L. supina was dissolved in 90% aqueous MeOH and partitioned with hexanes and CH2Cl2, successively. The CH2Cl2-soluble portion was repeatedly subjected to silica gel, Sephadex LH-20, and Lichroprep RP-C18 gel column chromatography and semipreparative RP-HPLC, to afford 10 new (1−10) and five known (11−15) labdane diterpenoids. Lagopsin A (1) was obtained as colorless plates via crystallization from MeOH, [α]21D +20. The positive-ion HRESIMS gave a pseudomolecular ion at m/z 401.2314 [M + Na]+ that together with 13C NMR spectroscopic data was consistent with a molecular formula of C22H34O5, indicating six indices of hydrogen deficiency. The IR spectrum showed absorption bands for hydroxy (3450 cm−1) and ester (1720 cm−1) functionalities. The 1H NMR data (Table 1) displayed singlets for three tertiary methyl groups at δH 1.00 (6H, s, CH3 × 2), and 1.27 (3H, s), one secondary methyl group at δH 1.12 (3H, d, J = 6.5 Hz), and resonances of three olefinic protons at δH 7.35 (1H, br s), 7.23 (1H, br s), and 6.27 (1H, br s). The 13 C NMR data of 1 showed 22 carbon resonances comprising an ester carbonyl (δC 172.4), a quaternary sp2 (δC 125.2), three quaternary sp3 (δC 78.3, 43.5, 33.9), three sp2 methine (δC Received: February 11, 2014 Published: April 8, 2014 1047
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142.9, 138.5, 110.7), four sp3 methine (δC 72.8, 72.4, 46.0, 38.3), five sp3 methylene (δC 43.7, 35.1, 34.1, 21.5, 18.6), and five methyl (δC 33.6, 23.8, 21.7, 19.5, 11.0) carbons. The 1D and 2D NMR spectroscopic data revealed that compound 1 belongs to the labdane class of diterpenoids possessing a furan moiety.15,17,18 The 1H and 13C NMR spectroscopic data of 1 were comparable to those of the semisynthetic 15,16epoxylabda-13(16),14-diene-6β,7β,9α-triol (12),14 except for the presence of resonances of an acetoxy group (δH 2.10, δC 172.4, 21.7) in 1. The deshielded H-6 resonance (δH 5.59, br s) indicated that the acetoxy group is located at C-6, as confirmed by the HMBC correlation between H-6 and the acetoxy carbonyl carbon. The NOESY data revealed that the relative configurations at C-5, -6, -7, -8, -9, and -10 of 1 are the same as those of 15,16-epoxylabda-13(16),14-diene-6β,7β,9α-triol (12). The absolute configuration of 1 was defined as (5S,6S,7R,8R,9R,10S) by single-crystal X-ray diffraction analysis (Figure 1). Therefore, the structure of 1 was established as (5S,6S,7R,8R,9R,10S)-6β-acetoxy-15,16-epoxylabda-13(16),14diene-7β,9α-diol, and this compound has been named lagopsin A. Lagopsin B (2) was obtained as a colorless gum, and its molecular formula was determined as C24H36O6 by the 13C NMR spectroscopic data and the HRESIMS showing a pseudomolecular ion at m/z 443.2416 [M + Na]+. The 1D Table 1. 1H NMR Data of Compounds 1−10 (δ in ppm, J in Hz, 500 MHz, in CDCl3) position
1a
1a 1b 2a 2b 3a 3b 5
1.47, m 1.47, m 1.62, m 1.51, m 1.35, m 1.19, m 1.75, d (1.5)
1.47, 1.47, 1.62, 1.51, 1.36, 1.21, 1.80,
6 7
5.59, br s 3.65, dd (11.0, 3.5) 2.07, m 1.86, m 1.84, m 2.56, m 2.50, m 6.27, br s
5.67, br s 4.90, dd (11.0, 3.5) 2.29, m 1.91, m 1.85, m 2.58, m 2.50, m 6.29, br s
8 11a 11b 12a 12b 14a
2a m m m m m m br s
14b 15
7.35, br s
7.37, br s
16a
7.23, br s
7.24, br s
17
1.12, d (6.5)
0.99, d (6.5)
18 19 20 22 23 24
1.00, 1.00, 1.27, 2.10,
1.00, 0.98, 1.29, 2.08,
16b
a
s s s s
s s s s
3/4a 1.36, m 1.28, m 1.62, m 1.53, m 1.35, m 1.19, m 1.57, d (1.5)/1.53, d (1.8) 5.60, t (3.0) 3.62,dd (11.0, 3.5)/ 3.58, dd (11.0, 3.5) 2.09, m 2.12, m 1.80, m 2.25, m/2.10, m 2.25, m/2.00, m 2.48, dd (13.5, 5.5)/ 2.16, dd (13.5, 5.5) 2.02, m/2.30, dd (13.5, 1.5) 5.56, d (5.0)/5.43, d (3.5) 3.98, d (8.5)/4.18, d (8.5) 3.79, d (8.5)/3.53, d (8.5) 1.04, d (7.0)/1.06, d (7.0) 0.99, s 0.98, s/0.99, s 1.22, s/1.24, s 2.11, s
5/6a 1.47, 1.42, 1.66, 1.52, 1.36, 1.17, 1.58,
m m m m m m br s/1.41, br s
5.61, br s 3.62, dd (11.0, 3.5)/ 3.67, dd (11.0, 3.5) 2.11, m 2.20, m 1.80, m 2.21, m/2.19, m 2.21, m/1.90, m 2.36, m/2.42, m
7a 1.47, 1.42, 1.65, 1.52, 1.36, 1.20, 1.77,
m m m m m m br s
8a 1.45, 1.37, 1.60, 1.50, 1.47, 1.32, 2.77,
m m m m m m d (3.0)
9a 1.50, 1.37, 1.63, 1.52, 1.46, 1.32, 2.73,
m m m m m m d (3.0)
10a 1.98, 1.66, 1.77, 1.65, 1.55, 1.40,
m m m m m m
5.61, br s 3.66, dd (11.0, 3.0) 2.06, m 1.82, t (8.0) 1.82, t (8.0) 2.44, m 2.44, m 6.79, br s
5.39, d (3.0)
5.38, d (2.5)
6.19, s
2.55, 2.75, 2.75, 2.60, 2.60, 6.27,
2.58, 2.84, 2.84, 2.53, 2.47, 7.19,
2.27, 1.92, 2.74, 2.67, 6.32,
5.75, br s
7.32, br s
m, 2.46, m m m m m br s
m, 2.44, m m m m m br s
m m m m br s
1.93, m/1.91, m 5.61, br s/5.42, d (4.0) 4.05, d (8.5)/4.27, d (9.0) 4.00, d (8.5)/3.74, d (9.0) 1.01, d (6.0)/1.00, d (6.0) 0.99, s 0.98, s 1.23, s/1.26, s 2.11, s
4.77, br s (2H)
7.22, br s
7.36, br s 7.27, br s
1.11, d (6.5)
1.05, t (7.5)
1.05, t (7.5)
1.44, s
1.00, 1.00, 1.25, 2.12, 3.59,
1.00, 1.05, 1.37, 2.06,
1.03, 1.06, 1.43, 2.09,
1.24, s 1.21, s 1.33, s
s s s s s
s s s s
s s s s
1.99, s
Assignments were based on HSQC and HMBC experiments. 1048
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confirmed by the HMBC correlation between H-7 (δH 4.90, dd, J = 11.0, 3.5 Hz) and the acetoxy carbonyl carbon (δC 170.5). Thus, the structure of 2 (lagopsin B) was identified as 6β,7βdiacetoxy-15,16-epoxylabda-13(16),14-diene-9α-ol. Lagopsin C (3) and 15-epi-lagopsin C (4) were obtained as an inseparable mixture (1:2), [α]21D −17. The positive-ion HRESIMS data showed a pseudomolecular ion at m/z 419.2402 [M + Na]+ and, in conjunction with the 13C NMR data, established a molecular formula of C22H36O6, implying five indices of hydrogen deficiency. The IR spectrum of 3/4 indicated the presence of hydroxy (3440 cm−1) and ester carbonyl (1736 cm−1) functional groups. The 1H and 13C NMR data (Tables 1 and 2) showed duplicate resonances for three tertiary methyl groups (δH 0.98/0.99, 0.99 × 2, 1.22/1.24; δC 19.8/19.9, 33.0/33.1, 23.9 × 2, respectively), one secondary methyl group (δH 1.04/1.06; δC 11.6/11.8), two oxygenated quaternary carbons (δC 93.2/93.7, 89.2/90.3), a hemiacetal methine (δH 5.56/5.43; δC 98.3/98.5), an isolated oxygenated methylene (δH 3.98/4.18, 3.79/3.53; δC 76.8/75.8), two oxygenated methines (δH 5.60 × 2, 3.62/3.58; δC 72.7/72.5, 73.2/73.1, respectively), six methylenes (δC 47.3/46.5, 44.0/ 43.9, 38.3/35.9, 34.1/34.3, 30.0/29.7, 18.6 × 2), two methines (δC 47.4/47.5, 38.4/38.2), and two quaternary carbons (δC 42.8 × 2, 34.0/34.2). These data suggested that compounds 3/4 are bis-spirolabdane diterpenoids possessing a 1,6-dioxaspiro[4.4]nonane C/D ring moiety.17,19−21 In addition, duplicate resonances of an acetoxy group (δH 2.11 × 2; δC 172.5/ 172.4, 21.7 × 2) were evident in the NMR spectra. Comparison of NMR data of 3/4 with those of lagopsin A (1) suggested that they share the same A- and B-ring substitution patterns. In turn, comparison of the 13C NMR chemical shifts with those of bis-spirolabdane diterpenoids15,19−21 supported the 1,6dioxaspiro[4.4]nonane moiety of 3/4. The molecular structures
Figure 1. ORTEP drawing of compound 1 (ORTEP diagram is one of two independent molecules of compound 1).
NMR data (Tables 1 and 2) of 2 were similar to those of 1, except for the presence of an acetoxy rather than a hydroxy group in 2. The C-7 location of the acetoxy group was
Table 2. 13C NMR Data of Compounds 1−11 (δ in ppm, 125 MHz, in CDCl3) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 a
1a 34.1, 18.6, 43.7, 33.9, 46.0, 72.4, 72.8, 38.3, 78.3, 43.5, 35.1, 21.5, 125.2, 110.7, 142.9, 138.5, 11.0, 33.6, 23.8, 19.5, 172.4, 21.7,
CH2 CH2 CH2 C CH CH CH CH C C CH2 CH2 C CH CH CH CH3 CH3 CH3 CH3 C CH3
2a 34.0, 18.6, 43.6, 34.0, 46.0, 69.2, 74.3, 36.3, 78.3, 43.5, 35.0, 21.5, 125.0, 110.7, 143.1, 138.5, 10.8, 33.5, 23.7, 19.4, 170.5, 21.5, 170.5, 20.9,
CH2 CH2 CH2 C CH CH CH CH C C CH2 CH2 C CH CH CH CH3 CH3 CH3 CH3 C CH3 C CH3
3/4a
5/6a
7a
34.1/34.3, CH2 18.6/18.6, CH2 44.0/43.9, CH2 34.0/34.2, C 47.4/47.5, CH 72.7/72.5, CH 73.2/73.1, CH 38.4/38.2, CH 93.2/93.5, C 42.8/42.8, C 30.0/29.7, CH2 38.3/35.9, CH2 89.5/90.3, C 47.3/46.5, CH2 98.3/98.5, CH 77.8/76.3, CH2 11.6/11.8, CH3 33.0/33.1, CH3 23.9/23.9, CH3 19.8/19.9, CH3 172.5/172.4, C 21.7/21.7, CH3
34.2/34.4, CH2 18.7/18.7, CH2 44.1/44.2, CH2 34.1/34.4, C 47.3/48.1, CH 72.9/72.4, CH 73.1/72.8, CH 38.3/38.3, CH 93.3/93.5, C 42.8/42.8, C 30.0/29.8, CH2 38.7/35.0, CH2 89.1/90.6, C 47.9/45.4, CH2 99.3/99.6, CH 78.4/77.4, CH2 11.6/11.8, CH3 33.0/33.2, CH3 23.8/23.8, CH3 19.8/19.9, CH3 172.3/172.3, C 21.7/21.7, CH3
34.1, 18.6, 43.6, 34.0, 46.1, 72.3, 72.9, 38.5, 77.9, 43.7, 32.3, 21.9, 139.0, 141.7, 102.7, 171.6, 10.9, 33.6, 23.8, 19.4, 172.4, 21.7, 57.1,
CH2 CH2 CH2 C CH CH CH CH C C CH2 CH2 C CH2 CH C CH3 CH3 CH3 CH3 C CH3 CH3
8a 39.3, 18.2, 41.8, 34.2, 47.7, 77.4, 208.3, 32.7, 215.0, 51.4, 37.7, 19.0, 124.3, 111.1, 142.6, 139.0, 7.4, 33.5, 24.6, 19.9, 170.0, 20.9,
CH2 CH2 CH2 C CH CH C CH2 C C CH2 CH2 C CH CH CH CH3 CH3 CH3 CH3 C CH3
9a 39.8, 18.2, 42.0, 34.6, 47.7, 77.3, 208.3, 32.7, 214.7, 51.5, 34.3, 19.9, 133.1, 145.5, 70.1, 174.2, 7.4, 33.6, 24.5, 19.9, 169.9, 20.9,
CH2 CH2 CH2 C CH CH C CH2 C C CH2 CH2 C CH CH2 C CH3 CH3 CH3 CH3 C CH3
10a 31.8, 17.1, 38.5, 37.1, 178.6, 121.4, 201.8, 78.3, 78.0, 47.3, 30.5, 20.8, 125.6, 111.0, 142.8, 138.6, 24.4, 32.0, 31.0, 27.0,
CH2 CH2 CH2 C C CH C C C C CH2 CH2 C CH CH CH CH3 CH3 CH3 CH3
11a 34.1, 18.7, 43.4, 34.3, 47.3, 68.7, 77.6, 35.8, 78.5, 43.3, 34.9, 21.4, 125.2, 110.8, 143.0, 138.5, 11.0, 33.7, 24.6, 19.6, 170.2, 21.2,
CH2 CH2 CH2 C CH CH CH CH C C CH2 CH2 C CH CH CH CH3 CH3 CH3 CH3 C CH3
Assignments were based on HSQC and HMBC experiments. 1049
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its 13C NMR and HRESIMS data (m/z 447.2359 [M + Na]+). The IR spectrum showed absorptions characteristic of hydroxy (3439 cm−1) and ester carbonyl (1766 and 1728 cm−1) groups. The 1H NMR data (Table 1) displayed singlets for three tertiary methyl groups at δH 1.00 (6H, s, CH3 × 2) and 1.25 (3H, s) and one secondary methyl group at δH 1.11 (3H, d, J = 6.5 Hz), indicative of 7 being a labdane-type diterpenoid.15,22,23 The 1H and 13C NMR data (Tables 1 and 2) were comparable to those of 1, with similar resonances for the A and B rings. The 13 C NMR resonances at δC 171.6, 141.7, 139.0, 102.7, and 57.1 and the 1H NMR resonances at δH 6.79 (1H, br s), 5.75 (1H, br s), and 3.59 (3H, s) indicated that 7 possesses a 3substituted 5-methoxyfuran-2(5H)-one moiety at C-12 rather than the furan ring of 1. A comparison of the NMR spectroscopic data of the C-11−C-16 region to those reported for the structural analogues sibiricinone C22 and 6β-acetoxy-9αhydroxy-15-methoxy-13(14)-labden-16,15-olide24 facilitated assembly of its C-11−C-16 component. The molecular structure of 7 was confirmed by the HMBC correlations between H3-20 and C-1/C-5/C-9; H3-18 and C-4/C-5/C-19; H3-17 and C-7/ C-8/C-9; H-15 and C-13/C-16/C-23; H-14 and C-13/C-15/ C-16; H2-12 and C-13/C-14; H2-11 and C-8/C-9/C-10/C-12/ C-13; H-8 and C-6/C-7/C-17; H-6 and C-5/C-7/C-8/C-10; and H3-23 and C-15 (Figure 3). The relative configurations at
of 3/4 were confirmed by the HMBC correlations between H320 and C-1/C-5/C-9; H3-18 and C-4/C-5/C-19; H3-17 and C7/C-8/C-9; H2-16 and C-12/C-15; H-15 and C-13/C-14/C16; H2-14 and C-12/C-13/C-16; H2-11 and C-8/C-10/C-13; and H-6 and C-5/C-7/C-8/C-10 (Figure 2). The location of
Figure 2. Selected HMBC (arrows point from protons to carbons) and NOE correlations of compounds 3/4.
the acetoxy group at C-6 was confirmed by the HMBC correlation between H-6 (δH 5.60, br s) and the acetoxy carbonyl carbon (δC 172.5/172.4). In the NOESY spectrum, the NOEs from H3-20 to H3-19 and from H-5 to H3-18 supported the trans A/B junction in 3/4. NOEs between H-5/ H-6, H-5/H-1α, H-6/H-7, and H-7/H3-17 indicated that these hydrogens are cofacial and α-oriented, while correlations between H3-20/H-8, H2-11, and H-8/H2-11 suggested that these protons are β-cofacially oriented. The NOE correlations between H2-14/H3-17 and H-1α/H2-16 suggested the relative C-13 configuration as shown in 3/4 (Figure 2). This observation also proved the α-orientation of Me-17. Furthermore, the NOE correlation between H-15 and H2-12 suggested the α-orientation of the HO-15 in 4, while the absence of a similar correlation indicated a β-oriented 15-OH group in 3. Accordingly, the structures of lagopsin C (3) and 15-epi-lagopsin C (4) were established as shown. An inseparable 7:10 epimeric mixture of lagopsin D (5) and 15-epi-lagopsin D (6) was obtained as a colorless gum, [α]21D −7. The IR spectrum exhibited absorption bands for hydroxy (3440 cm−1) and ester carbonyl (1735 cm−1) groups. The molecular formula C22H36O6 of 5/6 was determined to be the same as that of 3/4 from the 13C NMR and positive-ion HRESIMS data (m/z 419.2414 [M + Na]+). Slight differences in the 1H and 13C NMR spectroscopic values (Tables 1 and 2) of 5/6 in comparison with those of 3/4 suggested that they could be stereo- or regioisomeric compounds. 2D NMR experiments facilitated the assembly of the molecular structures of 5/6 as the same as those of 3/4. The relative configurations at C-5, -6, -7, -8, -9, and -10 were the same as assigned for 3/4 on the basis of NOESY data. The NOE correlations between H2-16/H3-17 and H-1α/H2-14 in compounds 5/6 in contrast to H2-14/H3-17 and H-1α/H2-16 in 3/4 indicated their regioisomeric nature. The respective α- and β-orientations of the 15-OH in 6 and 5 were assigned via the NOE correlation between H-15 and H2-12 in 6 and the absence of a similar association in 5. Therefore, the structures of lagopsin D (5) and 15-epi-lagopsin D (6) were defined as shown in 5/6. In view of the exposure of hemiacetals 3/4 and 5/6 to methanol, it is surprising that the corresponding acetals were absent. Lagopsin E (7) was isolated as a colorless gum, [α]21D +13, and its molecular formula was determined to be C23H36O7 via
Figure 3. Selected HMBC (arrows point from protons to carbons) correlations of compounds 7 and 8.
C-5, -6, -7, -8, -9, and -10 as assigned via NOE association were the same as determined for 1. However, the C-15 relative configuration could not be assigned. Thus, the structure of 7 was defined as 6β-acetoxy-7β,9α-dihydroxy-15-methoxy13(14)-labden-16,15-olide, named lagopsin E. Lagopsin F (8) was obtained as a colorless, viscous oil, [α]21D +9. Its 13C NMR and positive-ion HRESIMS data, exhibiting a sodiated molecular ion at m/z 399.2143, corresponded to a molecular formula of C22H32O5 (calcd for C22H32O5Na, 399.2142), with seven indices of hydrogen deficiency. The IR spectrum showed absorption bands at 1743, 1729, and 1698 cm−1, reminiscent of the presence of the ester and keto carbonyl functionalities. The 1H and 13C NMR spectra exhibited resonances for three tertiary methyl groups (δH 1.00, 1.05, 1.37; δC 33.5, 24.6, 19.9, respectively), a propanoyl group [δH 1.05 (3H, t, J = 7.5 Hz), 2.55, 2.46 (each 1H, m); δC 7.4, 32.7, 208.3], an acetoxy group (δH 2.06; δC 170.0, 20.9), a carbonyl group (δC 215.0), a β-substituted furan (δH 7.32, 7.22, 6.27; δC 111.1, 124.3, 139.0, 142.6), five methylenes, two methines (one oxygenated), and two quaternary carbons. These spectroscopic data indicated 8 to be an 8,9-seco-labdane diterpenoid with an acetoxy substituent.25,26 The 1D NMR data of 8 resembled those of the dioxo derivative of hispanolone26 except for the resonances of an acetoxy group in 8. The acetoxy group was assigned to C-6 via the HMBC correlation between H-6 and the acetoxy carbonyl carbon. The proposed structure of 8 was further confirmed by the HMBC 1050
dx.doi.org/10.1021/np5001329 | J. Nat. Prod. 2014, 77, 1047−1053
Journal of Natural Products
Article
By comparing spectroscopic and specific rotation data with literature values, the remaining three known compounds were identified as leoleorin A (13),15 leoheterin (14),14,16 and leoleorin B (15).15 Compounds 1−15 were evaluated for their inhibitory effects on the NO production in LPS-stimulated BV-2 microglial cells using the Griess assay.27,28 Compounds 7, 9, 13, and 15 showed moderate inhibitory activity against NO production with IC50 values of 16.2, 34.9, 14.9, and 34.0 μM, respectively. Quercetin was used as a positive control (IC50 = 10.0 μM) (Table 3). Compounds 1−6, 8, 10−12, and 14 were inactive
correlations between H3-20 and C-1/C-5/C-9/C-10; H3-18 and C-19/C-4/C-5; H3-17 and C-7/C-8; H-16 and C-13/C14/C-15; H-15 and C-13/C-14/C-16; H-14 and C-13/C-15/ C-16; H2-12 and C-9/C-11/C-13/C-14/C-16; H2-11 and C-9/ C-12/C-13; H-8 and C-7/C-17; and H-6 and C-4/C-5/C-7/C10 (Figure 3). The NOE correlation between H-6 and H3-20 indicated that CH3-20 and the C-6 side chain are cofacial. However, the C-6 relative configuration could not be assigned. Therefore, the structure of lagopsin F (8) was identified as shown. This compound may be derived from the 7-oxo derivative of compound 1 via a retro-aldol reaction with concomitant cleavage of the C-8−C-9 bond. Lagopsin G (9) was obtained as a colorless, viscous oil, [α]21D +15. Its molecular formula was established as C22H32O6 based on the 13C NMR data and a protonated molecular ion [M + H]+ at m/z 393.2256 (calcd for C22H33O6, 393.2272) in its HRESIMS. The IR spectrum exhibited absorption bands for ester (1744 cm−1) and keto carbonyl (1730, 1698 cm−1) functional groups. Comparison of their NMR data suggested that compound 9 differed from compound 8 by replacement of the β-substituted furan ring by a 3-substituted furan-2(5H)-one moiety [δH 4.77 (2H, br s), 7.19; δC 70.1, 133.1, 145.5, 174.2)],23 which was supported by the HMBC correlations between H-15 and C-13/C-14; H-14 and C-13; and H-12 and C-9/C-11/C-13/C-14/C-16. The relative configurations of C-5 and C-10 were the same as those of 8 based on the NOESY data, but the C-6 relative configuration could not be assigned. Accordingly, the structure of lagopsin G (9) was established as shown. Lagopsin H (10) was obtained as a white, amorphous powder, [α]25D −67, and its 13C NMR and positive-ion HRESIMS data showing a pseudomolecular ion at m/z 355.1879 [M + Na]+ established a molecular formula of C20H28O4, indicating seven indices of hydrogen deficiency. The IR spectrum indicated the presence of hydroxy (3431 cm−1) and α,β-unsaturated carbonyl (1660 cm−1) functionalities. The 1 H NMR data (Table 1) displayed singlets for four tertiary methyl groups at δH 1.44, 1.33, 1.24, and 1.21 and the resonances of four olefinic protons at δH 7.36 (1H, br s), 7.27 (1H, br s), 6.32 (1H, br s), and 6.19 (1H, s). The 13C NMR spectrum showed 20 carbon resonances including four methyls, five methylenes, four olefinic methines, and seven quaternary carbons (one carbonyl, two olefinic, and two oxygenated). These spectroscopic data revealed that compound 10 is a labdane diterpenoid with a furan moiety and an α,β-unsaturated carbonyl group.15 The 1D NMR data of 10 (Table 1) were nearly superimposable on those of leoleorin A (13), previously isolated from Leonotis leonurus,15 except for the significantly deshielded C-8 (δC 78.3; ΔδC +30.4) and C-17 (δC 24.4; ΔδC +14.5) resonances. This is in agreement with the addition of a C-8 hydroxy group in 10, which was supported by the presence of a methyl singlet in 10 instead of a doublet for H3-17 in 13. Thus, the structure of 10 (lagopsin H) was defined as 15,16epoxy-8β,9α-dihydroxylabda-5,13(16),14-trien-7-one. Compounds 11 and 12 were earlier reported as intermediates 7β-acetoxy-15,16-epoxylabda-13(16),14-diene-6β,9αdiol (11) and 15,16-epoxylabda-13(16),14-diene-6β,7β,9α-triol (12) in the syntheses of sibiricinone A and leoheterin.14 This is the first report of their natural occurrence. Owing to their structural similarity to compounds 1−10, the trivial names lagopsins I and J were assigned to 11 and 12. The specific rotation and 13C NMR data of lagopsin I (11) are presented herein for the first time.
Table 3. Inhibitory Effects of Compounds from L. supina on LPS-Activated NO Production in BV-2 Microglial Cells compounda 7 9 13 15 quercetinc
IC50 (μM)b 16.2 34.9 14.9 34.0 10.0
± ± ± ± ±
3.2 4.5 2.1 3.8 2.0
a
Compunds 1−6, 8, 10−12, and 14 were inactive (
