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Sep 8, 2015 - Pharmacy, University of Mississippi, University, Mississippi 38677-1848, United States. •S Supporting Information. ABSTRACT: Twenty ne...
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Anti-inflammatory Labdane Diterpenoids from Leonurus macranthus Zheng Huang,†,‡ Zhi-Xiang Zhu,† Yue-Ting Li,†,‡ Dao-Ran Pang,†,‡ Jiao Zheng,† Qian Zhang,† Yun-Fang Zhao,† Daneel Ferreira,§ Jordan K. Zjawiony,§ Peng-Fei Tu,† and Jun Li*,† †

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 § Department of BioMolecular Sciences, Division of Pharmacognosy, and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677-1848, United States

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S Supporting Information *

ABSTRACT: Twenty new polyoxygenated labdane diterpenoids (1−20) were isolated from the aerial parts of Leonurus macranthus. Their structures were elucidated on the basis of spectroscopic and spectrometric data (1D and 2D NMR, IR, and HRESIMS). The absolute configurations of macranthin A (1) and 6-O-deacetylmacranthin A (2) were determined by single-crystal X-ray crystallographic analysis and a modified Mosher’s method, respectively. Compounds 1−9, 12, 14, and 19 showed inhibition of nitric oxide production in lipopolysaccharide-activated BV-2 microglial cells with IC50 values of 10.0− 63.7 μM.

T

he genus Leonurus (family: Lamiaceae, syn. Labiatae) comprises approximately 25 species and five varieties, which are mainly distributed in the North Temperate Zone, particularly in Asia and Europe. Among them, 12 species, one variety, and two forma grow in mainland China.1,2 The dried aerial parts of many Leonurus species, commonly called Chinese motherwort (“yimucao” in Chinese), have been used in traditional Chinese medicine for the treatment of menoxenia, dysmenorrhea, amenorrhea, lochia, edema, oliguresis, and ulcerations.2,3 Previous phytochemical investigations of Leonurus species have indicated the presence of diterpenoids, alkaloids, flavonoids, iridoid glycosides, phenylethanoid glycosides, triterpenoids, cyclic peptides, megastigmanes, and sesquiterpenoids.3−5 A broad spectrum of pharmacological effects including cardioprotective, neuroprotective, hepatoprotective, antioxidative, cytotoxic, anti-inflammatory, analgesic, antiplatelet aggregation, estrogen sulfotransferase inhibitory, cholinesterase inhibitory, and antibacterial activities, as well as effects on the uterus, have been reported for crude extracts and pure compounds from Leonurus plants.3,4,6 A continued search for bioactive diterpenoids from medicinal plants7−10 afforded 20 new labdane diterpenoids (1−20) from the aerial parts of Leonurus macranthus Maxim. Herein, the isolation and structural elucidation of the isolates as well as their inhibitory effects on lipopolysaccharide (LPS)-activated nitric oxide (NO) production in BV-2 microglial cells are described.



was repeatedly subjected to silica gel, Sephadex LH-20, and Lichroprep RP-C18 gel column chromatography (CC) and semipreparative RP-C18 HPLC to afford 20 new labdane diterpenoids (1−20). Macranthin A (1) was obtained as colorless plates via crystallization from MeOH, [α]21 D −59. Its molecular formula, C26H40O9, was deduced from the HRESIMS (m/z 514.2997 [M + NH4]+, calcd for C26H44O9N, 514.3011) and 13C NMR spectroscopic data, indicating seven indices of hydrogen deficiency. The IR spectrum showed absorption bands for hydroxy (3562 cm−1) and carbonyl (1755, 1739, 1725 cm−1)

RESULTS AND DISCUSSION

The acetone extract of the aerial parts of L. macranthus was dissolved in 90% aqueous MeOH and partitioned with nhexane and CH2Cl2, successively. The CH2Cl2-soluble portion © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 20, 2015

A

DOI: 10.1021/acs.jnatprod.5b00635 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. 1H NMR Data of Compounds 1−10 (δ in ppm, J in Hz, 500 MHz) 1a

position 1a 1b 2a 2b 3 5

2.21, m 1.21, dt (9.5, 3.0) 2.03, m 1.70, m 3.80, br s 2.42, d (2.5)

6

5.17, d (2.5)

2a

3a

4a

5a

6a

7a

2.14, m 1.15, m

1.96, m 1.19, m

1.94, m 1.17, m

1.95, m 1.18, m

2.00, m 1.21, m

2.33, m 1.19, m

2.07, m 1.20, d (12.5)

2.05, m 1.66, m 3.77, br s 2.19, d (3.0) 4.12, d (2.5)

1.88, m 1.79, m 5.00, br s 2.41, d (3.0) 5.25, d (3.0)

1.87, m 1.79, m 5.00, br s 2.31, d (2.0) 5.18, s

1.89, 1.80, 5.00, 2.18,

1.96, 1.83, 5.20, 2.31,

1.92, 1.70, 3.76, 3.40,

1.93, 1.73, 5.02, 2.26,

m m br s m

4.24, d (2.0)

m m br s br s

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8

3.44, q (7.0)

11a 11b 12a 12b

2.30, 1.90, 1.87, 1.76,

m m m m

14a 14b 15a 15b 16 17

1.96, 1.93, 4.18, 4.08, 1.27, 1.03,

m m m m s d (6.5)

18 19a

1.06, s 4.46, d (11.5)

19b

4.42, d (11.5)

20 OAc-3 OAc-6 OAc-7 OAc-15 OAc-19

1.45, s

3.63, q (6.5) 2.26, m 1.93, m 1.87, m 1.74, m 1.95, m 1.86, m 4.17, m 4.07, m 1.26, s 1.05, d (7.0) 1.14, s 4.84, d (12.0) 4.60, d (12.0) 1.45, s

2.12, s 2.02, s 2.03, s

2.01, s 2.03, s

3.26, q (6.5) 2.15, m 1.83, m 1.83, m 1.68, t (10.0) 1.95, m 1.82, m 4.13, m 4.07, m 1.19, s 1.03, d (6.5) 1.01, s 4.39, d (12.0) 4.23, d (12.0) 1.40, s 2.02, s 2.09, s 1.99, s 2.02, s

3.29, q (6.5) 2.15, m 1.81, m 1.83, m 1.73, m

3.60, q (6.5) 2.18, m 1.87, m 1.86, m 1.76, m

3.33, q (6.0) 2.21, m 1.86, m 1.86, m 1.79, m

1.96, m 1.72, m 3.76, m 3.62, m 1.20, s 1.05, d (6.5) 0.98, s 4.39, d (12.0) 4.22, d (11.5) 1.40, s 2.08, s 2.00, s

2.00, m 1.70, m 3.82, m 3.66, m 1.24, s 1.10, d (6.5) 1.05, s 4.81, d (12.0) 4.49, d (12.0) 1.46, s 2.03, s

2.05, m 1.76, m 3.85, m 3.69, m 1.26, s 1.11, d (6.5) 1.08, s 4.01, d (11.5) 3.73, d (11.5) 1.43, s 2.08, s 2.10, s

2.01, s

2.06, s

9a

10a

2.02, m 1.16, d (11.5) 1.97, m 1.72, m 4.99, br s 2.07, m

2.01, m 1.14, d (12.5)

5.58, t (3.0)

4.23, m

4.09, t (2.5)

3.92, d (11.0) 1.83, m

3.58, dd (11.0, 3.5) 2.07, m

4.80, m 2.40, m

3.42, dd (11.0, 3.5) 2.10, m

2.13, 1.81, 1.95, 1.82,

m m m m

2.20, 1.80, 1.90, 1.76,

m m m m

2.20, 1.82, 1.90, 1.75,

m m m m

2.17, 1.78, 1.91, 1.74,

m m m m

2.11, m 1.97, m 4.23, m 4.23, m 1.37, s 1.18, d (7.0) 1.16, s 4.92, d (11.0) 4.38, d (11.0) 0.84, s

2.09, 1.92, 4.27, 4.23, 1.30, 1.04,

m m m m s d (6.5)

2.09, m 1.91, m 4.29, m 4.22, m 1.29, s 0.94, d (6.5) 1.04, s 4.89, d (12.0) 4.56, d (12.0) 1.29, s 2.07, s

2.09, 1.93, 4.25, 4.25, 1.28, 1.05,

m m m m s d (7.0)

2.05, s 2.04, s

2.11, s 2.03, s

m m br s s

5.23, br s

7

8a

m m br s d (2.0)

1.06, s 4.39, d (11.5) 4.11, d (11.5) 1.29, s 2.07, s 2.03, s

2.08, s 2.04, s 2.05, s

1.96, 1.71, 5.00, 2.04,

m m br s d (2.0)

1.07, s 4.87, d (12.0) 4.59, d (12.0) 1.27, s 2.06, s

2.05, s 2.03, s

a Assignments were based on HSQC and HMBC experiments. Compounds 1, 2, and 8−10 were measured in methanol-d4, and 3−7 were measured in CDCl3.

functionalities. The 1H NMR data (Table 1) displayed characteristic resonances of four methyl [δH 1.06, 1.27, 1.45 (each 3H, s); 1.03 (3H, d, J = 6.5 Hz)], two oxygenated methylene [δH 4.08 (1H, m), 4.18 (1H, m); δH 4.42 (1H, d, J = 11.5 Hz), 4.46 (1H, d, J = 11.5 Hz)], two oxygenated methine [δH 3.80 (1H, br s); δH 5.17 (1H, d, J = 2.5 Hz)], and three Oacetyl [δH 2.02, 2.03, 2.12 (each 3H, s)] groups. The 13C NMR data (Table 2) of 1 showed 26 carbon resonances due to seven methyl (δC 10.0, 20.7, 21.0, 21.1, 21.3, 22.4, 27.4), seven sp3 methylene (δC 26.2, 28.4, 30.4, 40.0, 42.3, 63.0, 68.7), four sp3 methine (δC 46.2, 47.9, 70.7, 78.6), two sp3 quaternary (δC 44.4, 44.7), two oxygenated sp3 tertiary (δC 84.9, 98.4), one ketocarbonyl (δC 206.5), and three ester carbonyl (δC 171.3, 172.8, 172.9) carbons. These functionalities accounted for four out of the seven indices of hydrogen deficiency, and the remaining three thus required the presence of a tricyclic system in 1. The aforementioned data suggested that this compound is a highly oxygenated tricyclic spirolabdane diterpenoid with three O-acetyl groups.9,11 The deshielded proton and carbon chemical shifts of C-3 (δH 3.80; δC 70.7), C-6 (δH 5.17; δC 78.6), C-15 (δH 4.08, 4.18; δC 63.0), and C-19 (δH 4.42, 4.46; δC 68.7) indicated that these four sp3carbons are oxygenated. This deduction was verified by the HMBC correlations from

H2-1, H2-2, H-5, H3-18, and H2-19 to C-3; from H-6 to C-4, C5, C-7, C-8, and C-10; from H2-15 to C-13 and C-14; and from H-3, H-5, and H3-18 to C-19. The only ketocarbonyl group could be located at C-7 on the basis of the HMBC correlations from H-5, H-6, H-8, and H-17 to C-7 (Figure 1). The deshielded resonances of H-6 (δH 5.17), H2-15 (δH 4.08, 4.18), and H2-19 (δH 4.42, 4.46) indicated that the three O-acetyl groups are located at C-6, C-15, and C-19. This was confirmed by the HMBC correlations from H-6, H2-15, and H2-19 to the acetoxy carbonyl carbons at δC 171.3, 172.9, and 172.8, respectively. The relative configuration of 1 was established on the basis of the NOESY data (Figure 1). The NOE correlations from H3-20 to H2-19 and from H-5 to H3-18 indicated that the A/B rings are trans-fused. NOEs of H-5/H-6, H-5/H-1α, H-5/ H3-18, and H-6/H3-18 suggested that these hydrogens are cofacial and α-oriented, while the NOE correlations of H-3/H219, H2-19/H3-20, H3-20/H-8, H3-20/H2-11, and H-8/H2-11 were consistent with these protons being β-cofacially oriented. The NOE correlations between H2-14/H3-17 and H-1α/H3-16 suggested the relative C-13 configuration as shown in 1 (Figure 1). This observation also proved the α-orientation of Me-17. Furthermore, the absolute configuration of 1 was defined as (3R,4S,5S,6S,8S,9R,10S,13S) on the basis of single-crystal X-ray B

DOI: 10.1021/acs.jnatprod.5b00635 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 13C NMR Data of Compounds 1−10 (δ in ppm, 125 MHz)

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position

1a

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

28.4, 26.2, 70.7, 44.7, 46.2, 78.6, 206.5, 47.9, 98.4, 44.4, 30.4, 40.0, 84.9, 42.3, 63.0, 27.4, 10.0, 22.4, 68.7, 21.1,

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

OAc-6

171.3, C 21.3, CH3

2a 28.4, 26.5, 70.8, 44.7, 46.2, 76.3, 212.4, 46.6, 98.4, 44.1, 30.2, 40.1, 84.5, 42.4, 63.1, 27.4, 10.1, 22.7, 70.2, 21.2,

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

3a 27.8, 22.5, 73.2, 42.3, 45.3, 76.5, 203.2, 46.5, 96.5, 43.0, 29.2, 38.9, 83.7, 41.2, 61.5, 27.0, 9.7, 21.8, 66.7, 20.9, 170.3, 20.9, 169.2, 21.4,

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

4a 27.8, 22.4, 73.1, 42.3, 45.9, 76.9, 204.3, 46.5, 97.3, 43.0, 29.1, 39.5, 85.4, 44.5, 59.4, 26.9, 9.5, 21.8, 66.6, 21.1, 169.3, 21.3, 170.2, 20.8,

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

5a 27.8, 22.7, 73.7, 42.6, 45.7, 75.3, 210.3, 45.9, 97.7, 42.7, 28.9, 39.8, 85.4, 44.2, 59.7, 26.8, 9.7, 22.0, 67.9, 21.1, 170.5, 21.0,

6a

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

28.1, 22.7, 73.4, 44.4, 46.2, 77.4, 204.7, 46.6, 97.7, 43.3, 29.2, 39.9, 85.7, 44.6, 59.6, 27.0, 9.7, 21.8, 66.1, 21.1, 170.5, 21.3, 169.3, 21.5,

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

7a 25.5, 24.8, 69.5, 40.9, 52.3, 212.1, 77.7, 47.1, 92.4, 48.0, 29.0, 39.4, 83.9, 41.3, 61.9, 27.4, 13.7, 21.2, 66.3, 20.8,

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

8a 28.8, 23.3, 75.0, 42.9, 44.6, 73.6, 73.2, 39.4, 94.8, 43.5, 30.3, 40.5, 84.2, 42.5, 62.9, 27.8, 12.8, 22.5, 68.1, 21.1, 172.1, 21.1, 173.0, 21.0,

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

OAc-7 OAc-15 OAc-19

172.9, 20.7, 172.8, 21.0,

C CH3 C CH3

173.1, 20.9, 173.0, 20.8,

C CH3 C CH3

171.0, 21.1, 171.0, 21.2,

C CH3 C CH3

171.0, C 20.8, CH3

171.3, C 21.3, CH3

171.3, 21.2, 171.1, 21.1,

C CH3 C CH3

172.9, 21.9, 172.7, 21.0,

C CH3 C CH3

9a

10a

28.9, 23.5, 75.2, 43.4, 45.3, 68.1, 79.0, 36.6, 95.1, 43.3, 30.3, 40.4, 84.2, 42.5, 62.8, 27.7, 12.5, 22.8, 69.4, 21.3, 172.4, 21.1,

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

28.9, 23.6, 75.4, 43.2, 45.3, 70.8, 74.8, 38.7, 95.2, 43.2, 30.3, 40.5, 83.8, 42.5, 63.0, 27.8, 13.0, 22.8, 69.7, 21.3, 172.4, 21.1,

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

172.8, 21.1, 172.9, 20.8, 173.0, 21.0,

C CH3 C CH3 C CH3

172.9, 21.0, 173.0, 20.8,

C CH3 C CH3

a Assignments were based on HSQC and HMBC experiments. Compounds 1, 2, and 8−10 were measured in methanol-d4, and 3−7 were measured in CDCl3.

Figure 1. Selected HMBC (arrows point from protons to carbons) and NOE correlations of compound 1.

diffraction analysis (Figure 2), which was supported by the modified Mosher’s method (Figure 3). Thus, the structure of 1 was established as (3R,4S,5S,6S,8S,9R,10S,13S)-6,15,19-triacetoxy-9,13-epoxy-3-hydroxylabdan-7-one and was named macranthin A. Compounds 2−6 were assigned the molecular formulas of C24H38O8, C28H42O10, C26H40O9, C24H38O8, and C24H38O8, respectively, from their 13C NMR and HRESIMS data. The 1H and 13C NMR data (Tables 1 and 2) exhibited characteristic resonances of a highly oxygenated tricyclic spirolabdane diterpenoid skeleton like 1. These resonances included four methyls (C-16, -17, -18, and -20), two oxygenated methylenes (C-15 and C-19), two oxygenated methines (C-3 and C-6), two oxygenated sp3 tertiary carbons (C-9 and C-13), and a ketocarbonyl group (C-7). The main differences were that

Figure 2. ORTEP drawing of compound 1.

two O-acetyl groups were present at C-15/C-19 in 2, C-3/C-19 in 5, and C-3/C-6 in 6; three O-acetyl groups were located at C-3/C-6/C-19 in 4; and four O-acetyl groups were located at C-3/C-6/C-15/C-19 in 3. These assignments were corroborated by their HMBC spectra (Figures S11, S28, S33, S23, and S18, respectively, Supporting Information). The relative configurations of compounds 2−6 were the same as assigned C

DOI: 10.1021/acs.jnatprod.5b00635 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Macranthin B (7) was obtained as a colorless gum, [α]21 D −20. Its 13C NMR and positive-ion HRESIMS data, exhibiting a sodium adduct ion at m/z 477.2462, corresponded to a molecular formula of C24H38O8 (calcd for C24H38O8Na, 477.2459), the same as that of 6-O-deacetylmacranthin A (2). Analysis of the 1H and 13C NMR spectroscopic data (Tables 1 and 2) of 7 showed a close structural resemblance to 2. The major difference was that the ketocarbonyl and hydroxy groups were located at C-6 and C-7, respectively, in 7. This assignment was supported by the significantly deshielded singlet of H-5 (δH 3.40; ΔδH +1.21) and shielded H-8 resonance (δH 1.83; ΔδH −1.80), and verified by the HMBC correlations from H-5, H-7, and H-8 to C-6; from H-7 to C-8 and C-17; and from H3-17 to C-7, C-8, and C-9 (Figure S38, Supporting Information). The H-7 proton was assigned as α-oriented by the NOE correlation of H-5/H-7 in the NOESY spectrum. The relative configurations of the remaining stereocenters in 7 were the same as those of 2 on the basis of NOESY data (Figure S39, Supporting Information). Accordingly, the structure of 7 (macranthin B) was defined as 15,19-diacetoxy-9,13-epoxy-3α,7β-dihydroxylabdan-6-one. Macranthin C (8) was obtained as a colorless gum, [α]21 D −20. Its molecular formula was determined to be C28H44O10 via the 13C NMR and HRESIMS data (m/z 563.2810 [M + Na]+),

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Figure 3. Δδ (δS − δR) values (in ppm, data obtained in pyridine-d5) for the MTPA esters of compounds 1 and 2.

for 1 on the basis of their NOESY data (Figures S12, S19, S24, S29, and S34, respectively, Supporting Information). The absolute configuration of 2 was identical to that of 1 as determined by the modified Mosher’s method (Figure 3). Therefore, the structures of compounds 2−6 were elucidated, in turn, as (3R,4S,5S,6S,8S,9R,10S,13S)-15,19-diacetoxy-9,13epoxy-3,6-dihydroxylabdan-7-one (6-O-deacetylmacranthin A, 2), 3α,6β,15,19-tetraacetoxy-9,13-epoxylabdan-7-one (3-O-acetylmacranthin A, 3), 3α,6β,19-triacetoxy-9,13-epoxy-15-hydroxylabdan-7-one (isomacranthin A, 4), 3α,19-diacetoxy-9,13epoxy-6β,15-dihydroxylabdan-7-one (6-O-deacetylisomacranthin A, 5), and 3α,6β-diacetoxy-9,13-epoxy-15,19-dihydroxylabdan-7-one (19-O-deacetylsiomacranthin A, 6).

Table 3. 1H NMR Data of Compounds 11−20 (δ in ppm, J in Hz, 500 MHz) 11a

position

12a

13a

1a

2.10, m

2.17, m

1.99, m

1b 2a 2b 3 5

1.28, 1.98, 1.70, 3.67, 2.45,

1.78, m 2.00, m 1.75, m 3.66, br s 2.28, dd (11.0, 3.5) 2.52, m

6b 8

1.27 m 1.97, m 1.75, m 3.72, br s 2.47, dd (14.5, 3.0) 2.38, dd (14.0, 3.5) 2.29, d (14.0) 2.70, q (6.5)

11a 11b 12a 12b 14a 14b 15a 15b 16 17

2.22, 1.84, 1.85, 1.74, 1.97, 1.86, 4.14, 4.10, 1.27, 1.04,

18 19a

1.06, s 4.20, d (11.0)

19b

3.98, d (11.5)

20 OAc-3 OAc-6 OAc-15 OAc-19

1.14, s

6a

m m m m m m m m s d (6.5)

2.02, s 2.05, s

m m m m m

2.46, m 2.29, m 2.91, q (6.5) 2.27, m 1.89, m 1.88, m 1.76, m 1.89, m 1.78, m 3.67, m 3.61, m 1.29, s 1.05, d (7.0) 1.02, s 4.28, d (11.5) 4.04, d (11.5) 1.19, s

2.04, s

14a 1.90, dt (13.0, 3.0) 1.68, m 2.14, m 1.69, m 3.80, br s 2.11, d (3.0) 4.16, d (3.0)

15a

16a

17a

18a

19a

20a

1.79, m

1.96, m

1.90, m

1.74, m

1.76, m

2.28, m

1.73, m 2.06, m 1.81, m 4.94, br s 2.08, d (2.5) 4.11, d (2.5)

1.75, 2.12, 1.72, 3.83, 2.44,

1.68, m 2.00, m 1.84, m 5.01, br s 2.35, d (3.5) 5.71, d (3.0)

1.68, m 2.02, m 1.86, m 5.09, br s 2.35, d (3.0) 5.73, d (3.5)

1.80, m 2.88, m 2.50, m

5.68, d (3.5)

1.68, m 2.05, m 1.76, m 3.90, br s 2.45, d (3.5) 5.72, d (3.0)

2.43, 2.43, 1.67, 1.67, 1.78, 1.78, 3.75, 3.73, 1.28, 1.84,

2.45, 2.45, 1.71, 1.64, 1.76, 1.76, 3.74, 3.74, 1.26, 1.81,

2.40, 2.40, 1.68, 1.62, 1.87, 1.87, 4.27, 4.26, 1.29, 1.82,

2.40, 2.40, 1.69, 1.66, 1.85, 1.70, 3.97, 3.91, 1.30, 1.83,

2.41, 2.41, 1.67, 1.67, 1.87, 1.87, 4.26, 4.26, 1.29, 1.84,

2.45, 2.45, 1.63, 1.63, 1.83, 1.70, 3.99, 3.92, 1.31, 1.84,

m m m br s m

2.24, d (3.0) 5.70, d (3.0)

2.45, m

2.39, 2.39, 1.68, 1.63, 1.78, 1.78, 3.74, 3.74, 1.25, 1.76,

m m m m m m m m s s

2.42, 2.42, 1.70, 1.62, 1.77, 1.77, 3.74, 3.74, 1.25, 1.82,

m m m m m m m m s s

1.04, s 4.30, d (11.5)

1.15, s 4.78, d (12.0)

4.09, d (11.5)

4.65, d (11.5)

1.14, s

1.43, s

2.05, s

m m m m m m m m s s

1.07, s 4.21, d (11.5) 3.58, d (11.5) 1.50, s 2.05, s

2.04, s

m m m m m m m m s s

m m m m m m m m s s

1.09, s 4.40, d (11.5) 4.32, d (11.5) 1.40, s

1.16, s 4.43, d (11.5) 4.15, d (11.5) 1.39, s

2.10, s

2.12, s 2.04, s 2.07, s

2.04, s

m m m m m m m m s s

1.05, s 4.42, d (11.5) 4.13, d (11.5) 1.40, s 2.07, s 2.11, s 2.05, s

m m m m m m m m s s

1.07, s 4.44, d (11.5) 4.14, d (11.5) 1.41, s 2.08, s 2.13, s 2.06, s 2.06, s

m m m m m m m m s s

1.27, s 4.89, d (11.5) 3.96, d (11.5) 1.61, s 2.11, s 2.00, s

a Assignments were based on HSQC and HMBC experiments. Compounds 11 and 17−20 were measured in CDCl3, and 12−16 were measured in methanol-d4.

D

DOI: 10.1021/acs.jnatprod.5b00635 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 4. 13C NMR Data of Compounds 11−20 (δ in ppm, 125 MHz)

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

11a 25.8, 25.5, 70.7, 42.4, 41.3, 38.9, 210.1, 50.6, 96.5, 41.8, 29.4, 39.2, 83.7, 41.2, 61.8, 27.3, 10.0, 22.1, 67.3, 18.7,

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

12a 27.2, 26.6, 71.0, 43.0, 43.3, 39.8, 214.0, 51.8, 98.3, 43.6, 30.2, 40.4, 85.4, 46.1, 60.0, 27.5, 10.3, 22.7, 68.4, 18.7,

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

13a 29.6, 26.6, 70.5, 42.3, 45.1, 35.7, 201.9, 130.9, 171.1, 42.1, 25.0, 41.8, 73.0, 43.9, 59.5, 26.3, 11.5, 22.3, 68.4, 18.3,

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

14a 31.2, 26.2, 70.4, 43.7, 48.6, 71.3, 200.0, 129.2, 171.7, 42.2, 25.4, 41.9, 73.1, 44.0, 59.5, 26.6, 11.8, 22.3, 69.6, 23.0,

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

15a 32.7, 24.2, 75.2, 44.4, 50.1, 70.9, 199.3, 129.1, 170.9, 42.4, 25.4, 41.9, 73.1, 44.0, 59.4, 26.6, 11.9, 21.9, 66.3, 22.1, 172.2, 21.0,

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

OAc-6 OAc-15 OAc-19

171.3, 21.2, 171.2, 21.1,

C CH3 C CH3

172.9, C 20.8, CH3

172.8, C 20.7, CH3

173.0, C 20.8, CH3

16a

17a

30.8, CH2 25.9, CH2 70.4, CH 43.6, C 47.7, CH 71.5, CH 194.7, C 130.2, C 172.2, C 41.9, C 25.9, CH2 41.9, CH2 73.0, C 44.0, CH2 59.4,CH2 26.6, CH3 11.8, CH3 21.7, CH3 67.9, CH2 23.0, CH3

29.8, CH2 25.2, CH2 70.0, CH 42.4, C 46.5, CH 70.1, CH 192.7, C 129.6, C 168.6, C 40.7, C 24.3, CH2 41.1, CH2 72.0, C 40.0, CH2 61.2,CH2 26.6, CH3 11.8, CH3 22.7, CH3 66.8, CH2 22.7, CH3

171.4, C 21.5, CH3

169.8, 21.6, 171.1, 21.2, 171.0, 21.0,

172.7, C 20.6, CH3

C CH3 C CH3 C CH3

18a 30.6, 22.6, 72.5, 41.5, 47.9, 69.6, 192.5, 129.6, 168.7, 40.6, 24.4, 41.2, 73.4, 41.5, 59.9, 26.3, 11.8, 21.3, 66.0, 22.6, 170.4, 21.2, 169.8, 21.6,

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

171.1, C 20.9, CH3

19a 30.7, CH2 22.6, CH2 72.5, CH 41.5, C 48.0, CH 69.6, CH 192.5, C 130.0, C 168.1, C 40.7, C 24.3, CH2 41.1, CH2 72.0, C 40.0, CH2 61.2,CH2 26.6, CH3 11.8, CH3 21.3, CH3 66.0, CH2 22.6, CH3 170.3, C 21.3, CH3 169.7, C 21.6, CH3 171.1, C 21.0, CH3 171.1, C 21.2, CH3

20a 36.8, 35.0, 210.1, 51.8, 53.4, 70.2, 192.3, 129.9, 166.6, 40.7, 24.6, 41.0, 73.2, 41.8, 60.0, 26.4, 11.9, 20.8, 66.3, 21.5,

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

169.5, C 22.3, CH3

171.0, C 22.3, CH3

a

Assignments were based on HSQC and HMBC experiments. Compounds 11 and 17−20 were measured in CDCl3, and 12−16 were measured in methanol-d4.

S49, Supporting Information). Thus, the structure of 9 (isomacranthin C) was defined as 3α,7β,15,19-tetraacetoxy9,13-epoxy-6β-hydroxylabdane. The molecular formula of compound 10 was determined as C26H42O9 by the 13C NMR data and a sodium adduct ion at m/ z 521.2709 [M + Na]+ in the positive-ion HRESIMS, which is 42 mass units less than that of macranthin C (8), suggesting it is a deacetylated derivative of 8. The 1H and 13C NMR spectroscopic data (Tables 1 and 2) of 10 were similar to those of 8, with the difference being the replacement of the C-6 acetoxy group in 8 by a hydroxy group in 10. The molecular structure of 10 was confirmed by the HMBC spectrum (Figure S53, Supporting Information). The relative configuration of 10 was the same as that of 8 on the basis of NOESY data (Figure S54, Supporting Information). Accordingly, the structure of 10 (6-O-deacetylmacranthin C) was established as 3α,15,19triacetoxy-9,13-epoxy-6β,7β-dihydroxylabdane. Macranthin D (11) gave a molecular formula of C24H38O7 based on the 13C NMR data and a protonated ion at m/z 439.2678 [M + H]+ (calcd for C24H39O7, 439.2690) in the positive-ion HRESIMS. Analysis of the 1H and 13C NMR spectroscopic data (Tables 3 and 4) of 11 showed a close structural resemblance to 6-O-deacetylmacranthin A (2). The major difference involved replacement of the C-6 hydroxymethine function [δH 4.12 (1H, d, J = 2.5 Hz); δC 76.3] in 2 by a methylene group [δH 2.38 (1H, dd, J = 14.0, 3.5 Hz), 2.29 (1H, d, J = 14.0 Hz); δC 38.9] in 11. The relative configurations at C-3, -4, -5, -8, -9, -10, and -13 were the same as assigned for 2 on the basis of NOESY data (Figure S59, Supporting Information). Therefore, the structure of 11 (macranthin D)

indicating the molecular mass of 8 to be two mass units more than that of 3-O-acetylmacranthin A (3). The 1H and 13C NMR spectroscopic data (Tables 1 and 2) of 8 were similar to those of 3, implying that these compounds share the same carbon skeleton. Interpretation of its 1D and 2D NMR data revealed that the C-7 ketocarbonyl function (δC 203.2) in 3 is replaced by a hydroxymethine group [δH 3.58 (dd, J = 11.0, 3.5 Hz); δC 73.2] in 8. This deduction was confirmed by the HMBC correlations from H-5 to C-6 and C-7; from H-6 to C-4, C-5, C-7, C-8, and C-10; from H-7 to C-6, C-8, and C-17; and from H3-17 to C-7, C-8, and C-9 (Figure S43, Supporting Information). In the NOESY spectrum (Figure S44, Supporting Information), NOE correlations of H-5/H-6, H-5/H-7, and H7/H3-17 indicated these hydrogens are cofacial and α-oriented. Therefore, the structure of 8 (macranthin C) was characterized as 3α,6β,15,19-tetraacetoxy-9,13-epoxy-7β-hydroxylabdane. Compound 9 shared a molecular formula, C28H44O10, with macranthin C (8) as determined by the 13C NMR data and a sodium adduct ion at m/z 563.2816 [M + Na]+ in the positiveion HRESIMS. The 1H and 13C NMR spectroscopic data (Tables 1 and 2) were comparable to those of 8, except for the significantly shielded H-6 (δH 4.23; ΔδH −1.35) and C-6 (δC 68.1; ΔδH −5.5) resonances, as well as the deshielded H-7 (δH 4.80; ΔδH +1.22) and C-7 (δC 79.0; ΔδC +5.8) resonances. This is in agreement with the OH-6 and OAc-7 substitutions in 9, which were supported by the HMBC correlations from H-7 to C-8, C-17, and the carbonyl carbon (δC 172.8) of an O-acetyl group and from H3-17 to C-7, C-8, and C-9 (Figure S48, Supporting Information). The relative configuration of 9 was the same as assigned for 8 on the basis of NOESY data (Figure E

DOI: 10.1021/acs.jnatprod.5b00635 J. Nat. Prod. XXXX, XXX, XXX−XXX

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HMBC correlations from H2-19 to the acetoxy carbonyl carbon (δC 172.8). The chemical shift of C-13 (δC 73.0) and the HMBC correlations from H2-15 and H3-16 to C-13 suggested that C-13 is hydoxylated. In the NOESY spectrum, the NOEs from H3-20 to H2-19 and from H-5 to H3-18 were supportive of a trans A/B-ring junction in 13. NOE cross-peaks of H-5/H-1α and H-5/H3-18 indicated that these hydrogens are cofacial and α-oriented, while NOEs between H-3/H2-19 and H2-19/H3-20 suggested that these protons occupy the opposite side and are thus β-oriented (Figure 4). However, the C-13 absolute configuration could not be assigned. Therefore, the structure of 13 (macranthin E) was defined as 19-acetoxy-3α,13,15trihydroxylabdan-8(9)-en-7-one. Macranthin F (14) had a molecular formula of C22H36O7 as determined by the 13C NMR data and a protonated molecular ion at m/z 413.2547 [M + H]+ (calcd for C22H37O7, 413.2534) in the positive-ion HRESIMS, which was 16 mass units more than that of 13. The 1H and 13C NMR data (Tables 3 and 4) of 14 were similar to those of 13, with the difference being due to the presence of resonances of an additional hydroxy group in 14. The hydroxy group was located at C-6 by the chemical shift of H-6 (δH 4.16) and C-6 (δC 71.3), as well as the HMBC correlations from H-6 to C-5, C-7, and C-10 (Figure S73, Supporting Information). In the NOESY spectrum, NOE correlations of H-6/H-5 and H-6/H3-18 indicated the αorientation of H-6 (Figure S74, Supporting Information). However, the absolute configuration at C-13 could not be assigned. Thus, the structure of 14 was established as 19acetoxy-3α,6β,13,15-tetrahydroxylabdan-8(9)-en-7-one. Compounds 15−19 gave the molecular formulas of C22H36O7, C24H38O8, C26H40O9, C26H40O9, and C28H42O10, respectively, as determined by the 13C NMR and HRESIMS data. Their 1H and 13C NMR data (Tables 3 and 4) exhibited characteristic resonances of a highly oxygenated labdane diterpenoid skeleton similar to 14, including four methyls (C16, -17, -18, and -20), two oxygenated methylenes (C-15 and C-19), two oxygenated methines (C-3 and C-6), one oxygenated sp3 tertiary carbon (C-13), and an enone system (C-7−C-9). The main differences were that an O-acetyl group is present at C-3 in 15; two O-acetyl groups were located at C6/C-19 in 16; three O-acetyl groups were linked to C-6/C-15/ C-19 in 17 and C-3/C-6/C-19 in 18; and four O-acetyl groups were located at C-3/C-6/C-15/C-19 in 19. These assignments were verified by their HMBC spectra (Figures S78, S83, S88, S93, and S98, respectively, Supporting Information). The relative configurations at C-3, -4, -5, -6, and -10 of compounds 15−19 were the same as those of 14 on the basis of their NOESY data (Figures S79, S84, S89, S94, and S99, respectively, Supporting Information). However, the absolute configuration at C-13 in compounds 15−19 could not be assigned. Accordingly, the structures of compounds 15−19 were defined as 3α-acetoxy-6β,13,15,19-tetrahydroxylabdan-8(9)-en-7-one (isomacranthin F, 15), 6β,19-diacetoxy-3α,13,15-trihydroxylabdan-8(9)-en-7-one (6-O-acetylmacranthin F, 16), 6β,15,19triacetoxy-3α,13-dihydroxylabdan-8(9)-en-7-one (6,15-diacetylmacranthin F, 17), 3α,6β,19-triacetoxy-13,15-dihydroxylabdan8(9)-en-7-one (3,6-diacetylmacranthin F, 18), and 3α,6β,15,19tetraacetoxy-13-hydroxylabdan-8(9)-en-7-one (3,6,15-triacetylmacranthin F, 19). Macranthin G (20) was obtained as a colorless gum, [α]21 D −39. Its molecular formula was determined as C24H36O8 from the 13C NMR and positive-ion HRESIMS data (m/z 453.2481 [M + H]+), indicating the molecular mass of 20 to be two mass

was defined as 15,19-diacetoxy-9,13-epoxy-3α-hydroxylabdan7-one. Compound 12 gave a molecular formula of C22H36O6 on the basis of the 13C NMR data and a protonated ion at m/z 397.2569 [M + H]+ (calcd for C22H37O6, 397.2585) in the positive-ion HRESIMS, which was 42 mass units less than that of 11, suggesting it to be a deacetylated derivative of macranthin D (11). The 1H and 13C NMR spectroscopic data (Tables 3 and 4) of 12 were comparable to those of 11, with the difference being the replacement of the C-15 acetoxy group in 11 by a hydroxy group in 12. The NOESY data revealed that 12 had the same relative configuration as 11 (Figure S64, Supporting Information). Thus, the structure of 12 was determined as 19-acetoxy-9,13-epoxy-3α,15-dihydroxylabdan-7-one, and this compound was named 15-Odeacetylmacranthin D. Macranthin E (13) was isolated as a colorless gum, [α]21 D +31, and its 13C NMR and positive-ion HRESIMS data showed a protonated ion at m/z 397.2566 [M + H]+, to establish a molecular formula of C22H36O6, indicating five indices of hydrogen deficiency. The IR spectrum exhibited the presence of hydroxy (3383 cm−1), ester carbonyl (1732 cm−1), and α,βunsaturated carbonyl (1661 cm−1) functionalities. The 1H NMR data (Table 3) displayed resonances of four methyl [δH 1.04, 1.14, 1.25, and 1.76 (each 3H, s)], two oxygenated methylene [δH 4.09 (1H, d, J = 11.5 Hz), 4.30 (1H, d, J = 11.5 Hz); δH 3.74 (2H, m)], one oxygenated methine [δH 3.66 (1H, br s)], and one O-acetyl [δH 2.05 (3H, s)] group. The 13C NMR spectrum showed 22 carbon resonances including five methyl, eight methylene (two oxygenated), two methine (one oxygenated), one ketocarbonyl, one ester carbonyl, two olefinic, one oxygenated secondary, and two quaternary carbons. These functionalities accounted for three of the five indices of hydrogen deficiency, requiring the presence of a bicyclic system in 13. The aforementioned data suggested that 13 is a highly oxygenated bicyclic labdane diterpenoid carrying an α,βunsaturated carbonyl moiety and sharing the same carbon skeleton with the 7-oxo-labd-8-en-15-ol isolated from Aeonium lindlryi.12 The assignment of the structure of 13 was facilitated by 2D NMR spectra and comparison with reported data. In the HMBC spectrum (Figure 4), the correlations from the proton

Figure 4. Selected HMBC (arrows point from protons to carbons) and NOE correlations of compound 13.

at δH 1.76 (H3-17) to the carbon resonances at δC 201.9, 171.1, and 130.9 confirmed the C-7−C-9 enone system. HMBC correlations from H-3 to C-1 and C-5 and from H3-18 and H219 to C-3 permitted the location of a hydroxy group at C-3. The acetoxy group was located at C-19 on the basis of the deshielded H2-19 resonances (δH 4.30, 4.09) as well as the F

DOI: 10.1021/acs.jnatprod.5b00635 J. Nat. Prod. XXXX, XXX, XXX−XXX

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semipreparative RP-HPLC column (YMC-Pack C18, 250 × 10 mm, 5 μm) was employed for the isolation. TLC was performed using precoated silica gel GF254 plates, sprayed with anisaldehyde reagent, prepared by mixing anisaldehyde (0.5 mL) with glacial HOAc (10 mL), MeOH (85 mL), and concentrated H2SO4 (5 mL), and heated until optimal color development. All purified compounds submitted for bioassay were at least 95% pure as judged by HPLC and supported by 1H NMR analyses. Plant Material. The aerial parts of L. macranthus were collected in Huanren, Liaoning Province, People’s Republic of China, in August 2013, and plant authentication was performed by one of the authors (P.-F.T.). A voucher specimen (JLI-LM-201308) is deposited in the Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine. Extraction and Isolation. The air-dried and powdered aerial parts of L. macranthus (5.0 kg) were extracted with acetone (3 × 30 L) by maceration at room temperature. After filtration and solvent evaporation, the residue (362.8 g) was dissolved in 90% aqueous MeOH and partitioned with n-hexane and CH2Cl2, successively. The dried CH2Cl2 residue (18.0 g) was subjected to VLC on silica gel eluting with an n-hexane−EtOAc solvent system with increasing polarity to afford nine fractions (A−I). Fraction C (2.37 g) was chromatographed over Sephadex LH-20 CC eluting with CH2Cl2− MeOH (1:1) to afford six fractions (C1−C6). Fraction C2 (1.54 g) was subjected to RP-C18 CC using a stepwise gradient of MeOH in H2O from 75% to 80% to give four fractions, C2a−C2d. Fraction C2b (915.0 mg) was chromatographed over silica gel CC eluting with nhexane−EtOAc (2:1) to afford 3 (148.5 mg). Fraction E (1.27 g) was subjected to Sephadex LH-20 CC eluting with CH2Cl2−MeOH (1:1) to afford nine fractions (E1−E9). Fraction E3 (260.2 mg) was purified via RP-C18 CC eluting with 75% MeOH in H2O followed by semipreparative RP-C18 HPLC using a mobile phase of isocratic MeOH−H2O (75:25) to afford 8 (12.7 mg, tR 25.0 min) and 9 (5.2 mg, tR 20.5 min). Separation of E4 (608.4 mg) by RP-C18 CC using a stepwise gradient of MeOH in H2O from 60% to 100% gave seven fractions, E4a−E4g. Fraction E4b (200.1 mg) was separated by silica gel CC using a mobile phase of n-hexane−EtOAc (2:1) to produce five subfractions, E4b1−E4b5. Fraction E4b2 (32.0 mg) was further purified by semipreparative RP-C18 HPLC with MeOH−H2O (53:47) as the mobile phase to afford 7 (11.1 mg, tR 14.5 min) and 11 (1.5 mg, tR 15.5 min). Compound 17 (1.2 mg, tR 25.0 min) was obtained from E4b5 by semipreparative RP-C18 HPLC using MeCN−H2O (35:65) as mobile phase. Fraction E4c was purified by semipreparative RP-C18 HPLC (isocratic 70% MeOH in H2O) to give 1 (174.6 mg, tR 15.5 min) and 2 (94.7 mg, tR 16.5 min). Fraction E4g (40.3 mg) was subjected to semipreparative RP-C18 HPLC using a mobile phase of MeOH−H2O (73:27) to yield 10 (1.3 mg, tR 25.8 min). Fraction F (2.30 g) was fractionated by Sephadex LH-20 CC eluting with CH2Cl2−MeOH (1:1) to afford five subfractions, F1−F5. Fraction F2 (1.31 g) was subjected to RP-C18 CC eluting with MeOH−H2O (from 50:50 to 100:0) to afford six fractions (F2a−F2f). Fraction F2b (279.4 mg) was subjected to silica gel CC eluting with n-hexane−EtOAc (2:1 → 1:1) to give five fractions, F2b1−F2b5. Compounds 4 (22.5 mg, tR 10.5 min), 6 (3.6 mg, tR 16.8 min), and 18 (6.7 mg, tR 8.8 min) were purified from fractions F2b3, F2b4, and F2b5 by semipreparative RPC18 HPLC eluting with isocratic 75%, 58%, and 75% MeOH in H2O, respectively. Fraction F2d (98.3 mg) was purified by silica gel CC and further by semipreparative RP-C18 HPLC using MeOH−H2O (73:27) as mobile phase to yield 19 (10.9 mg, tR 12.5 min). Fraction F3 (660.4 mg) was subjected to RP-C18 CC eluting with a stepwise gradient of MeOH in H2O from 40% to 80% to give nine fractions, F3a−F3h. Compound 5 (23.3 mg, tR 11.5 min) was purified by semipreparative RP-C18 HPLC with MeCN−H2O (46:54) from fraction F3f. Fraction G (1.31 g) was fractionated by RP-C18 CC (MeOH−H2O, 40:60 → 100:0) followed by semipreparative RP-C18 HPLC (isocratic 50% MeOH in H2O) to afford 20 (1.3 mg, tR 22.5 min). Fraction H (3.05 g) was repeatedly chromatographed on RP-C18 and silica gel CC and purified by semipreparative RP-C18 HPLC (isocratic 35% MeCN in H2O) to produce 12 (11.5 mg, tR 19.5 min). Fraction I (2.03 g) was subjected to RP-C18 CC eluting with a stepwise gradient of a MeOH−

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units less than that of 6-O-acetylmacranthin F (16). Comparison of their NMR data (Tables 3 and 4) suggested that compound 20 differs from compound 16 by replacement of the C-3 hydroxymethine function with a ketocarbonyl group (δC 210.1), which was supported by the HMBC correlations from H2-1, H2-2, H-5, H3-18, and H2-19 to C-3 (Figure S103, Supporting Information). The relative configurations at C-4, -5, -6, and -10 as assigned via NOE association were the same as determined for 16 (Figure S104, Supporting Information). Thus, the structure of 20 (macranthin G) was defined as 6β,19diacetoxy-13,15-dihydroxylabdan-8(9)-en-3,7-dione. Compounds 1−9, 12, 14−16, 18, and 19 were evaluated for their inhibitory activities against LPS-activated NO production in BV-2 microglial cells using the Griess assay. 13−16 Indomethacin served as a positive control, with an IC50 value of 35.7 μM. As shown in Table 5, compounds 1−9, 12, 14, and Table 5. Inhibitory Effects of Compounds from Leonurus macranthus on LPS-Activated NO Production in BV-2 Microglial Cells compounda 1 2 3 4 5 6 indomethacinc

IC50 (μM)b

compounda

± ± ± ± ± ± ±

7 8 9 12 14 19

63.7 45.7 48.1 20.7 41.7 16.2 35.7

7.1 1.4 5.8 5.0 9.6 3.9 1.3

IC50 (μM)b 13.2 11.0 10.0 36.9 53.6 12.7

± ± ± ± ± ±

2.0 4.0 4.5 2.3 11.2 4.7

a

Compounds 15, 16, and 18 were inactive (