Neglschisandrins A-B: two new dibenzocyclooctene lignans from Schisandra neglecta.

Two new dibenzocyclooctene lignans, neglschisandrins A-B (1-2), were isolated from the stems of Schisandra neglecta. Their structures and stereochemistries were elucidated by spectroscopic methods, including 1D- and 2D-NMR and HR-ESI-MS techniques.

Introduction

The stems or fruits of plants in the Schisandraceae family are widely used in China as tonic and astringent drugs for the treatment of rheumatic arthritis, traumatic injuries and related diseases [1]. Plants of the Schisandraceae are rich in lignans, especially dibenzocyclooctene lignans, which have been found to possess some beneficial effects such as anti-HIV effect, antitumor-promoting effect, calcium antagonism effect, and anti-lipid peroxidation effect, etc. [2,3,4,5,6]. In our previous studies, some new dibenzocyclooctene lignans from the Schisandraceae family were reported [7,8,9]. To search for new biological active natural products, another Schisandraceae plant, Schisandra neglecta, was investigated. Out examination of fractions extracted from the stems of S. neglecta with Et2O led to the isolation and identification of two new lignans, neglschisandrins A-B (1-2). This paper deals with the isolation and characterization of these new compounds.

Results and Discussion

Repeated column chromatography of the Et2O extract of the stem of S. neglecta yielded two new lignans, neglschisandrins A-B (1-2, Figure 1).

Figure 1

Structures of Compounds 1-2.

Neglschisandrin A (1), obtained as colorless powder, has the molecular formula C32H36O7, based on HR-ESI-MS data [m/z 555.2330 ([M+Na]+)]. The UV spectrum, with maximum absorptions at 220, 251 and 280 nm, along with corresponding 1H- and 13C-NMR spectra (Table 1) indicated that 1 is a dibenzocyclooctene lignan [10].

Table 1

NMR data of compounds 1-2 (400 MHz for 1H, 100 MHz for 13C) in CDCl3 (δ in ppm, J in Hz).

No	1		2
	δC	δH (Mult., J )	δC	δH (Mult., J )
1	151.2		151.1
2	139.4		139.8
3	153.0		153.2
4	107.5	6.48 (s)	107.2	6.60 (s)
5	140.1		137.7
6	35.5	6α: 2.33 (dd,13.4/9.6)	36.9	2.98 (d,12.6)
		6β: 2.06 (d,12.8)		2.98 (d,12.6)
7	40.5	1.80 (m)	154.0
8	33.8	1.94 (m)	38.7	2.70 (m)
9	39.2	9α: 2.64 (dd,13.7/7.4)	37.8	9α: 2.58 (m)
		9β: 2.56 (dd, 13.7/1.9)		9β: 2.58 (m)
10	133.9		133.1
11	113.1	6.72 (s)	112.8	6.73 (s)
12	151.5		151.8
13	139.6		139.9
14	142.3		142.4
15	123.6		124.1
16	120.9		121.2
17	21.6	1.00 (d, 7.1)	111.0	4.72 (d,1.5) 4.83 (d,1.5)
18	12.9	0.80 (d,7.1)	20.5	1.04 (d,7.2)
1-OMe	60.7	3.61 (s)	60.5	3.56 (s)
2-OMe	60.9	3.75 (s)	60.8	3.81 (s)
3-OMe	55.9	3.82 (s)	55.9	3.86 (s)
12-OMe	56.1	3.92 (s)	56.1	3.92 (s)
13-OMe	60.9	3.86 (s)	60.9	3.85 (s)
Cin: 1'	164.4
2'	117.3	6.37 (d,16.0)
3'	145.6	7.62 (d,16.0)
4'	134.3
5'/9'	128.1	7.44 (m)
6'/8'	128.8	7.34 (m)
7'	130.3	7.34 (m)
Ang: 1'			165.7
2'			127.8
3'			137.0	5.88 (m)
4'			15.3	1.78 (d,6.1)
5'			20.3	1.77 (s)

The 1H-NMR spectrum of 1 showed signals due to two Me groups at δH 1.00 and 0.80 (each 3H, d, J=7.1 Hz), assignable to the cis-oriented 7-Me and 8-Me, respectively [11]. The presence of four methylene signals (δH 2.33, 1H, dd, J=13.4, 9.6 Hz; 2.06, 1H, d, J=12.8 Hz; 2.56, 1H, dd, J=13.7, 1.9 Hz and 2.64, 1H, dd, J=13.7, 7.4 Hz) indicated that, like the known schisanhenol [12], 1 has no substitution at C-6 and C-9. Based on the HMQC spectrum, the protons at δH 2.33 and 2.06 were attached to the same carbon (δC 35.5). Similarly, the protons at δH 2.56 and 2.64 were attached to the same carbon at δC 39.2. Furthermore, HMBC correlations of δH 2.06 with δC 21.6 (C-17) and δC 40.5 (C-7) and of δH 2.56 with δC 12.9 (C-18) and δC 33.8 (C-8) indicated that δH 2.33 and 2.06 were H2-6 and that δH 2.56 and 2.64 were H2-9 (Figure 2).

Figure 2

Key HMBC Correlations of 1.

The 1H-NMR spectrum of 1 also showed signals due to two aromatic protons (δH 6.48, 6.72, each 1H, s) and five methoxy groups (δH 3.92, 3.86, 3.82, 3.75 and 3.61, each 3H, s) on two aromatic rings. The 1H-NMR spectrum also showed the presence of a trans-cinnamic acid ester, with proton signals at δH 6.37 and 7.62 (each 1H, d, J=16.0 Hz) and aromatic proton signals at δH 7.44 (2H, m) and 7.34 (3H, m). Carbon signals at δC 117.3, 145.6, 134.3, 128.1(x2), 128.8(x2) and 130.3, as well as carbonyl carbon at δC 164.4, supported this deduction [13].

HMBC correlations of δH 6.48 with δC 35.5 (C-6) and δH 6.72 with δC 39.2 (C-9) suggested that these two protons were H-4 and H-11, respectively. Their corresponding carbon signals were assigned as δC 107.5 and 113.1, respectively, by HMQC techniques. Based on HMBC correlations of H-4 with the aromatic carbons at δC 139.4, 151.2 and 153.0 and of H-11 with δC139.6, 142.3 and 151.5, these six carbons were assigned to C-2, -1, -3, -13, -14 and -12, respectively. The positions of the five methoxy substituents were elucidated from the HMBC cross peaks of δH 3.75, 3.61, 3.82, 3.86 and 3.92 with δC 139.4 (C-2), 151.2 (C-1), 153.0 (C-3), 139.6 (C-13) and 151.5 (C-12), respectively. Thus, the cinnamoxyl group should be located at the C-14 position (Figure 2).

The circular dichroism (CD) spectrum showed a negative Cotton effect at 216 nm and a positive Cotton effect at 249 nm, indicating that 1 has a R-biphenyl configuration [14]. The NOESY cross peaks (Figure 3) for H-4 with CH3-17, H-11 with H-9 and H-11 with CH3-18 in 1 suggested a twist-boat-chair (TBC) conformation for the cyclooctadiene ring [15]. The stereochemical assignments in the cyclooctadiene ring of 1 were supported by other NOESY correlations of H-4 with H-6β, CH3-17 with CH3-18, H-4 with 3-OMe, H-6β with H-7, H-11 with 12-OMe, and H-4 with H-7 (Figure 3). From the above data, the structure of 1 was elucidated as (6R, 7S, R-biar)- 3-phenyl-acrylic acid-2,3,10,11,12-pentamethoxy-6,7-dimethyl-5,6,7,8-tetrahydro -dibenzo[a,c]cycloocten-1-yl ester.

Figure 3

Key NOESY Correlations of 1.

Neglschisandrin B (2), obtained as colorless powder, has the molecular formula C28H34O7 according to HR-ESI-MS [m/z 505.2181 ([M+Na]+)]. The UV absorptions (216, 248 and 280 nm) and NMR spectra (Table 1) indicated that 2 was also a dibenzocyclooctene-type lignan. Its IR, UV, CD and NMR spectra were similar to those of 1. The differences between 2 and 1 were the substituent at C-14 and the formation of an exocyclic double bond between C-7 and C-17.

The 1H-NMR spectrum of 2 showed signals due to one Me group at δH 1.04 (3H, d, J=7.2 Hz), assignable to 8-Me. The presence of four methylene signals (δH 2.98, 2H, d, J=12.6 Hz; 2.58, 2H, m) indicated that 1 has no substitution at C-6 and C-9. Based on the HMQC spectrum, the protons at δH 2.58 (2H) were attached to the same carbon (δC 37.8) was well as the protons at δH 2.98 (2H) attached to the carbon at δC 36.9. Furthermore, HMBC correlations of δH 2.58 with δC 20.5 (C-18) and δC 38.7 (C-8) indicated that δH 2.58 (2H. m) were H2-9 (Figure 4). Then, the δH 2.98 (2H) were located at H2-6.

Figure 4

Key HMBC Correlations of 2.

The 1H-NMR spectrum of 2 also showed signals due to two aromatic protons (δH 6.60, 6.73, each 1H, s) on two aromatic rings. Their corresponding carbon signals were assigned as δC 107.2 and 112.8, respectively, by HMQC techniques. The chemical shifts of the protons and carbons indicated that they are located in H-4/11 and C-4/11 similar to the authentic sample [2]. HMBC correlations of δH 6.73 with δC 37.8 (C-9) suggested this proton was H-11. Therefore the another proton (δH 6.60) should be H-4, which was confirmed by the HMBC correlations of δH 6.60 with 36.9 (C-6).

Five methoxy groups (δH 3.56, 3.81, 3.86, 3.92 and 3.85, each 3H, s) on two aromatic rings were also observed from the 1H-NMR spectrum. An angeloyl group was observed in 1H-NMR (δ 5.88 (1H, m), 1.78 (3H, d, J=6.1 Hz), 1.77 (3H, s) and 13C-NMR data (δ 165.7, 127.8, 137.0, 15.3 and 20.3) [16]

Based on HMBC correlations of H-4 with the aromatic carbons at δC 139.8, 151.1 and 153.2 and of H-11 with δC 139.9, 142.4 and 151.8, these six carbons were assigned to C-2, C-1, C-3, C-13, C-14 and C-12, respectively (Figure 4). The positions of the five methoxy substituents were elucidated from the HMBC cross peaks of δH 3.81, 3.56, 3.86, 3.92 and 3.85 with δC 139.8 (C-2), 151.1 (C-1), 153.2 (C-3), 139.9 (C-11) and 151.8 (C-12), respectively. Thus, the angeloyl group was located at C-12 position.

The 13C NMR spectrum of 2 showed the presence of a quaternary olefinic carbon at δ 154.0 and an olefinic methylene carbon at 111.0, indicating the existence of the double bond. HMQC spectrum suggested the corresponding proton signal of δC 111.0 were assigned to δH 4.72 and 4.83 (each 1H, d, J=1.5 Hz). The correlations of δH 4.72, 4.83 with δ38.7 (C-8), 36.9 (C-6), and 154.0 based on HMBC suggested δC 111.0 and 154.0 were located at C-17 and at C-7, respectively (Figure 4), revealed the formation of an exocyclic double bond between C-7 and C-17, similar to that of the authentic sample of schisandrene reported in the literature [17].

The CD spectrum of 2 had a negative Cotton effect at 211 nm and a positive Cotton effect at 247 nm indicating that 2 has a R-biphenyl configuration. The NOESY correlations of H-4 with H-6β, H-4 with CH2-17, H-9α with CH3-18, and H-9α with H-11 in 2 suggested a twist-boat-chair (TBC) conformation for the cyclooctadiene ring (Figure 5). The substituent positions and stereochemical assignments in the cyclooctadiene ring of 2 were supported by other NOESY correlations of H-4/MeO-3, H-8/ CH2-17, H-6β/CH2-17, and H-11/CH3-18. Thus, the structure of 2 was determined as that shown in Figure 1.

Figure 5

Key NOESY Correlations of 2.

Experimental

General

TLC: Silica-gel plates GF254 (Yan-tai Institute of Chemical Technology). Column chromatography (CC): Silica gel (200-300 mesh or 300-400 mesh: Qingdao Marine Chemical Factory). Prep HPLC: Amersham UV-900, with RP-C18 column (250×10 mm). UV: Hitachi U-3010 spectrophotometer, in anh. MeOH; λmax in nm (log ε). CD Spectra: Jasco-810 spectropolarimeter; λ in nm (∆ε in mdeg). Optical rotation (ORD): JASCO P-1020 spectropolarimeter. IR Spectra: Avatar 360-ESP spectrophotometer (Thermo Nicolet), as KBr pellets; in cm-1. 1H-NMR and 13C-NMR spectra: Bruker DRX400 Spectrometer, in CDCl3 soln; δ in ppm rel. to Me4Si, J in Hz. HR-ESI-MS: Bruker Dalonics-BioToF Q spectrometer; in m/z.

Plant Material

The stems of Schisandra neglecta were collected in Lin-zhi County, Xi-zang Autonomous Region, People’s Repbulic of China in September of 2004, and identified by Associate Professor Hong-ping Deng of the School of Life Sciences, SouthWest University. A voucher specimen (MC-LZ-040901) is deposited in the Herbarium of Medicinal Plant, School of Life Sciences, SouthWest University, Chongqing, People’s Republic of China.

Extraction and Isolation

The air-dried stems (5 kg) of Schisandra neglecta were ground and extracted exhaustively with 95% ethanol at r. t. The EtOH extract was evaporated in vacuo to yield a semisolid (430 g), which was suspended in H2O (1 L) and extracted with Et2O (5×1L). This ether solution was concentrated to yield 112 g of residue, which was subjected to CC (SiO2, 1.5 kg, petroleum ether (PE)/acetone gradient.) Fr. 4 (eluted with PE/acetone 9:1) was subjected to repeated CC (eluted with PE/EtOAc 10:1) and prep. RP-HPLC (MeOH/H2O 75:25) to yield 1 (30 mg). Fr. 5 (eluted with PE/acetone 8:2) was subjected to repeated CC (eluted with PE/EtOAc 5:1) and prep. RP-HPLC (MeOH/H2O 75:25) to give 2 (46 mg).

Spectroscopic data for (6R,7S,R-biar)-3-phenylacrylic acid-2,3,10,11,12-pentamethoxy-6,7-dimethyl-5,6,7,8-tetrahydrodibenzo[a,c]cycloocten-1-yl ester (neglschisandrin A, 1). Colorless powder; UV (MeOH): 220 (4.80), 251 (4.48), 280 (4.47); CD (c=0.022, MeOH): nm (Δε) 249 (+13.76), 216 (‑23.86); =+40.2°(c=0.53, MeOH); IR (KBr): 3417, 2927, 1729, 1596, 1493, 1456, 765, 705; 1H-NMR and 13C-NMR: see Table 1; ESI-MS m/z: 555.4 ([M+Na]+); HR-ESI-MS: 555.2330 ([M+Na]+, C32H36O7Na; calc 555.2353).

Spectroscopic data for (7S,R-biar)-2-methylbut-2-enoic acid 2,3,10,11,12-pentamethoxy-6-methyl-7-methylene-5,6,7,8-tetrahydrodibenzo[a,c]cycloocten-1-yl ester (neglschisandrin B, 2). Colorless powder; UV (MeOH): 216 (4.21), 248 (3.77), 280 (3.26); CD (c=0. 0.088, MeOH): nm (Δε) 247 (+1.43), 236 (+1.68), 211 (-2.03); =+17.9°(c=0.11, MeOH); IR (KBr): 2933, 1734, 1597, 1491, 735; 1H-NMR and 13C-NMR: see Table 1; ESI-MS m/z: 505.3 ([M+Na]+); HR-ESI-MS: 505.2181 ([M+Na]+, C28H34O7Na, calc 505.2197).