- Email: [email protected]
Five new nervogenic acid derivatives from Liparis nervosa
Chinese Chemical Letters 24 (2013) 734–736
Contents lists available at SciVerse ScienceDirect
Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet
Original article
Five new nervogenic acid derivatives from Liparis nervosa Shuai Huang a,b, Ming-Feng Pan a, Xian-Li Zhou a,*, Zi-Li Zhou a, Cui-Juan Wang a, Lian-Hai Shan a, Jie Weng b,* a b
School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China Key Laboratory of Advanced Technology of Materials, Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 March 2013 Received in revised form 4 April 2013 Accepted 18 April 2013 Available online 4 June 2013
Five new nervogenic acid derivatives (1–5) have been isolated from Liparis nervosa (Thunb. ex A. Murray) Lindl. Their structures were elucidated on the basis of extensive spectroscopic analysis, including 1D NMR, 2D NMR, and HR-ESI-MS. ß 2013 Xian-Li Zhou and Jie Weng. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Orchidaceae Liparis nervosa Benzamide derivatives Nervogenic acid derivatives
2. Experimental
(1 L) at 50 8C and adjusted to pH 2.8 with 10% HCl, and extracted with CHCl3 and EtOAc to obtain the CHCl3 extract (82 g) and the EtOAc extract (25 g), respectively. The pH of the aqueous layer was adjusted to 9.4 with aqueous ammonia solution and extracted with EtOAc and n-butanol to afford the crude alkaline EtOAc extract (10 g) and the n-butanol extract (50 g), respectively. The n-butanol extract (50 g) was subjected to a column using D 101 resin and eluted with H2O, 30%, 60% and 95% EtOH, to afford four fractions (I, II, III, and IV). Fraction I (6.5 g) was subjected to RP-18 silica gel column, eluted in a step gradient manner with MeOH–H2O (1:50– 1:0) to afford fractions (A–F) based on TLC analysis. Fraction A (400 mg) was subjected to silica gel column, eluted with CHCl3:CH3OH:EtOAc (12:1:1) to yield compound 1 (50 mg), 2 (42 mg), 3 (23 mg), 4 (10 mg) and 5 (12 mg), respectively.
2.1. Plant material
2.3. Acid hydrolysis of 1–5
L. nervosa was collected in Chongqing, China in July 2007. The plant was identified by Professor Liangke Song in the School of Life Science and Engineering, Southwest Jiaotong University, Sichuan, China, where a voucher specimen is deposited (No. Z36150701).
Compound 1 (2 mg) was heated in 1 mol/L HCl–dioxane (1:1, 4 mL) at 80 8C for 4 h. After cooling, the solution was diluted with H2O (3 mL), neutralized with 1 mol/L NaOH and then extracted with CHCl3 (3 3 mL). The aqueous layer was concentrated under a stream of nitrogen. The residue was then dissolved in anhydrous pyridine (0.1 mL), followed by the addition of 0.1 mol/L L-cysteine methyl ester hydrochloride (0.2 mL). The resulting solution was stirred at 60 8C for 1 h, followed by the addition of trimethysilylation reagent hexamethyldisilazane–trimethylchlorosilane–pyridine (HMDS–TMCS–pyridine, 3:1:9). It was then stirred at 60 8C for an additional 30 min. The thiazolidine derivatives were analyzed by GC for sugar identification. GC analyses were performed using a Hewlett Packard GC6890 instrument on an
1. Introduction Liparis nervosa (Thunb. ex A. Murray) Lindl., a member of the Orchidaceae family, is an herbaceous plant widely distributed in China. It has been used in folk medicine for detoxification and hemostatic function [1]. Previous chemical investigations of L. nervosa led to the isolation of a series of nervogenic acid derivatives and pyrrolizidine alkaloids [2–5]. Our continued studies on the whole plant extract of L. nervosa have resulted in the isolation and structure determination of five new nervogenic acid derivatives (1–5) (Fig. 1).
2.2. Extraction and isolation The shade-dried L. nervosa (5.0 kg) was extracted with 95% EtOH (3 20 L). The EtOH extract (300 g) was suspended in H2O
* Corresponding authors. E-mail addresses: [email protected] (X.-L. Zhou), [email protected] (J. Weng).
1001-8417/$ – see front matter ß 2013 Xian-Li Zhou and Jie Weng. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. http://dx.doi.org/10.1016/j.cclet.2013.04.043
[(Fig._1)TD$IG]
S. Huang et al. / Chinese Chemical Letters 24 (2013) 734–736
O
b-D-glucopyranosyl units by gas chromatography of the hydro-
7 CR1
lyzed product and by the coupling constant of their anomeric protons Glc H-100 (d 4.74, d, J = 7.2 Hz) and Glc H-1000 (d 4.46, d, J = 7.8 Hz). In the HMBC experiment, long-range correlations were observed from Glc H-100 with C-4 (d 157.5) and Glc H-1000 with Glc C-400 (d 81.0). The linkage and the anomeric configuration of the sugar units agreed with the cellobiose unit of the nervosine III [5]. These data supported the structure of compound 1 as 3,5-bis(3methyl-2-butenyl)-4-O-[b-D-glucopyranosyl-(1!4)-b-D-glucopyranosyl] benzoic acid. Compound 2 was isolated as an amorphous solid. Its molecular formula was deduced as C28H40O12 by HR-ESI-MS at m/z 591.2417 [M+Na]+ (calcd. for C28H40O12Na: 591.2417). It was found to have a similar structure to compound 1 by comparison of their NMR data, the main difference observed was that a b-D-glucopyranoside unit was replaced by a a-L-arabinopyranose unit. It was confirmed by gas chromatography of the hydrolyzed product and by the coupling constant of their anomeric protons Ara H-100 (d 4.73, d, J = 7.6 Hz) and Glc H-1000 (d 4.77, d, J = 7.6 Hz). In the HMBC experiment, long-range correlations were observed from Ara H-100 with C-4 (d 158.1), Glc H-1000 with Ara C-200 (d 80.5). Moreover, the linkage of the sugar units was as same as in nervosine IV [5]. Thus, the structure of compound 2 was determined as 3,5-bis(3-methyl2-butenyl)-4-O-[b-D-glucopyranosyl-(1!2)-a-L-arabinopyranosyl] benzoic acid. Compound 3 was isolated as a white, amorphous solid and showed the molecular formula is C29H43NO12 by analysis of its HRESI-MS spectrum (m/z 620.2667 [M+Na]+, calcd. for C29H43NO12Na: 620.2683). The NMR spectroscopic data (Tables 1 and 2) of 3 were comparable to those of 1 and 2, indicating that 3 was also a nervogenic acid derivative. Comparison of the 13C NMR data (Table 2) of aglycon moiety of 3 with those of 3,5-bis(3methyl-2-butenyl)-4-O-(b-D-glucopyranosyl) benzamide [4], suggested that they possessed the same aglycone but differed in the sugar part. The sugar residues were identified as two Dglucopyranosides by gas chromatography. By analysis of the HMQC, HMBC and COSY data, compound 3 was determined to be 3,5-bis(3-methyl-2-butenyl)-4-O-[b-D-glucopyranosyl-(1!2)-bD-glucopyranosyl] benzamide.
R1
1 4'
3'
2'
3
1'
735
5'
5
6'
7'
OR2
8'
9'
10'
R2
1 OH
Glc ←1Glc
2 OH
Ara2←1Glc
3 NH2
Glc2←1Glc
4 NH2 5 NH2
4
Ara2←1Glc Glc4←1Glc
Fig. 1. Structures of compounds 1–5.
Agilent HP-5 column (0.25 mm, 30 m, i.d., 0.25 mm). Temperatures of both the injector and detector were 200 8C. A temperature gradient system was used for the oven, starting at 60 8C for 2 min and increasing up to 260 8C at a rate of 15 8C/min. D-Glucose and Larabinose were confirmed by comparison with the retention time of the authentic standards at 11.81 min and 9.04 min, respectively. The same analysis was carried out on compounds 2–5 [4]. 3. Results and discussion Compound 1 was isolated as a white, amorphous solid. Its molecular formula was determined as C29H42O13 by analysis of its HR-ESI-MS spectrum (m/z 599.2704 [M+H]+, calcd. for C29H43O13: 599.2706). The 1H NMR spectrum (Table 1) displayed a singlet at d 7.68 (s, 2H), indicating the presence of two aromatic protons. In addition, signals due to two prenyl groups were present, namely, d 5.31 (t, 2H, J = 7.2 Hz), d 3.59 (d, 4H, J = 7.2 Hz), and two singlets at d 1.75 (s, 6H) and d 1.77 (s, 6H), in total indicating 12 protons [4]. HMBC correlations were also observed from H-2 and H-6 (d 7.68) with the carbon resonance at dC 170.5 (C-7), indicating that the carboxyl group was attached to C-1. Comparison of the NMR data (Tables 1 and 2) of 1 with those published in literature [4], suggested that the aglycone moiety of 1 is 4-hydroxy-3,5-bis-(3methyl-2-butenyl)-benzoic acid, previously described as nervogenic acid unit [4]. The sugar residues were identified as two
Table 1 1 H NMR dataa of compounds 1–5. H
1b
2/6 10 /60 20 /70 40 /90 50 /100
7.68 3.59 5.31 1.75 1.77 Glc 4.74 3.59 3.60 3.68 3.29
100 200 300 400 500 600
10 00 20 00 30 00 40 00 50 00 60 00 a 1 b c
s d (7.2) t (7.2) s s d (7.2) m m m m
a 3.78 dd (2.0, 12.0) b 3.83 dd (6.4, 12.0) Glc 4.46 d (7.8) 3.26 m 3.35 m 3.31 m 3.36 m a 3.67 dd (5.6, 12.0) b 3.90 dd (2.0, 12.0)
H NMR (400 MHz, d, J in Hz). Data in CD3OD. Data in DMSO-d6.
2b
3b
7.66 s 3.54 d (7.2) 5.32 t (7.2) 1.73 s 1.76 s Ara 4.73 (7.6) 4.16 m 3.84 m 3.86 m a 3.40 overlapped b 3.90 dd (2.8, 8.4) –
7.67 3.45 5.21 1.67 1.71 Glc 4.81 3.82 3.43 3.40 3.46
Glc 4.77 d (7.6) 3.35 m 3.40 m 3.35 m 3.30 m a 3.67 dd (5.0, 11.3) b 3.80 dd (2.2, 11.3)
4c s d (7.2) t (7.2) s s overlapped m m m m
a 3.77 dd (7.0, 12.0) b 3.82 dd (2.2, 12.0) Glc 4.80 overlapped 3.31 m 3.14 m 3.30 m 3.43 m a 3.67 dd (5.2, 12.0) b 3.62 brd (12.0)
7.48 3.55 5.29 1.69 1.69 Ara 4.72 4.01 3.74 3.64 3.33 3.81 –
5c s d (7.6) t (7.6) s s d (5.2) m m m brd (11.6) dd (2.0, 11.6)
Glc 4.48 d (7.6) 3.02 m 3.17 m 3.10 m 3.40 m a 3.60 dd (5.2, 11.6) b 3.39 overlapped
7.48 3.54 5.27 1.70 1.70 Glc 4.65 4.04 3.72 3.54 3.21
s d (7.6) t (7.6) s s d (6.4) m m m m
a 3.47 overlapped b 3.72 brd (11.2) Glc 4.31 d (7.2) 3.00 m 3.16 m 3.07 m 3.30 m a 3.53 overlapped b 3.66 brd (11.8)
S. Huang et al. / Chinese Chemical Letters 24 (2013) 734–736
736 Table 2 13 C NMR dataa of compounds 1–5. C
1b
1c
2b
3b
4c
5c
1 2/6 3/5 4 7 10 /60 20 /70 30 /80 40 /90 50 /100
128.6 s 130.3 d 136.9 s 157.5 s 170.5 s 29.6 t 124.0 d 133.9 s 18.1 q 26.0 q Glc-I 105.7 d 76.3 d 75.4 d 81.0 d 76.7 d 62.0 t Glc-II 104.6 d 74.9 d 77.9 d 71.4 d 78.3 d 62.4 t
126.9 128.8 135.7 156.4 167.4 28.2 123.1 132.4 18.1 25.8 Glc-I 104.5 d 74.9 d 74.0 d 80.5 d 75.1 d 60.7 t Glc-II 103.3 d 73.5 d 76.7 d 70.3 d 77.1 d 61.3 t
127.9 s 130.3 d 137.0 s 158.1 s 169.9 s 29.6 t 123.8 d 134.0 s 18.1 q 26.0 q Ara 105.8 d 80.5 d 73.9 d 69.1 d 66.7 t – Glc 104.9 d 75.7 d 78.0 d 71.5 d 78.0 d 62.7 t
131.3 s 128.3 d 137.3 s 156.6 s 172.5 s 29.8 t 124.3 d 133.4 s 18.3 q 26.0 q Glc-I 104.3 d 82.9 d 78.1 d 71.4 d 78.4 d 62.7 t Glc-II 105.4 d 76.0 d 78.1 d 71.7 d 78.1 d 63.0 t
130.5 s 127.1 d 135.1 s 155.5 s 168.2 s 28.7 t 123.3 d 132.0 s 18.2 q 25.9 q Ara 104.4 d 79.5 d 71.4 d 66.4 d 64.6 t – Glc 103.1 d 74.4 d 76.8 d 70.2 d 77.1 d 61.2 t
130.7 s 127.1 d 135.3 s 154.9 s 168.1 s 28.4 t 123.5 d 131.8 s 18.1 q 25.9 q Glc-I 104.5 d 74.9 d 74.0 d 80.5 d 75.0 d 60.7 t Glc-II 103.3 d 73.5 d 76.7 d 70.3 d 77.0 d 61.2 t
100 200 300 400 500 600 100 0 200 0 300 0 400 0 500 0 600 0
the HMBC experiment. Thus the structure of compound 4 was determined as 3,5-bis(3-methyl-2-butenyl)-4-O-[b-D-glucopyranosyl-(1!2)-a-L-arabinopyranosyl] benzamide. The molecular formula of 5 was established as C29H43NO12 on the basis of the HR-ESI-MS. Comparison of the NMR data of compounds 5 and 4 (Tables 1 and 2) indicated that 5 possessed the same aglycone moiety. Complete assignment of the glycosidic protons and carbons along with an acid hydrolysis indicated the presence of two b-D-glucopyranosyl units, which has a (1!4) linkage by comparison of the NMR data with those of compound 1. By analysis of the HMQC, HMBC and COSY data, 5 was elucidated as 3,5-bis(3-methyl-2-butenyl)-4-O-[b-D-glucopyranosyl-(1!4)-bD-glucopyranosyl] benzamide. Acknowledgments This research work was financially supported by the National Natural Science Foundation of China (Nos. 21142004, 31171695), the New Century Talents Scheme of the Ministry of Education (No. NCET-08-0820), Science and Technology Support Programs of Sichuan Province (No. 2013SZ0083), and the Fundamental Research Funds for Central Universities (Nos. SWJTU2010ZT09, SWJTU12CX048).
a 13 b c
C NMR (100 MHz, d). Data in CD3OD. Data in DMSO-d6.
Compound 4 was isolated as an amorphous solid. Its molecular formula was deduced as C28H41NO11 by HR-ESI-MS at m/z 568.2744 [M+H]+ (calcd. for C28H42NO11: 568.2758). The HR-ESIMS and 13C NMR spectra (Table 2) revealed that compound 4 was also a benzamide derivative. After hydrolysis, sugars were identified as D-glucopyranoside and L-arabinopyranoside, in a ratio of 1:1, based on the GC analysis of their chiral derivatives. Their sequencing was identified as the same as in compound 2 by
References [1] Y.X. Hua, S.F. Liu, Z.Q. Yang, The Chinese Herbal, vol. 12, Shanghai Science and Technology Publishing House, Shanghai, 1999p. 997. [2] K. Nishikawa, Y. Hirata, Chemotaxonomical alkaloid studies. I. Structure of nervosine, Tetrahedron Lett. 27 (1967) 2591–2596. [3] K. Nishikawa, M. Miyamura, Y. Hirata, Chemotaxonomical alkaloid studies structures of Liparis alkaloids, Tetrahedron 25 (1969) 2723–2741. [4] S. Huang, X.L. Zhou, C.J. Wang, et al., New nervogenic acid derivatives from Liparis nervosa, Planta Med. 79 (2013) 281–287. [5] S. Huang, X.L. Zhou, C.J. Wang, et al., Pyrrolizidine alkaloids from Liparis nervosa with inhibitory activities against LPS-induced NO production in RAW264.7 macrophages, Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013. 03.009.
Contents lists available at SciVerse ScienceDirect
Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet
Original article
Five new nervogenic acid derivatives from Liparis nervosa Shuai Huang a,b, Ming-Feng Pan a, Xian-Li Zhou a,*, Zi-Li Zhou a, Cui-Juan Wang a, Lian-Hai Shan a, Jie Weng b,* a b
School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China Key Laboratory of Advanced Technology of Materials, Ministry of Education, Southwest Jiaotong University, Chengdu 610031, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 March 2013 Received in revised form 4 April 2013 Accepted 18 April 2013 Available online 4 June 2013
Five new nervogenic acid derivatives (1–5) have been isolated from Liparis nervosa (Thunb. ex A. Murray) Lindl. Their structures were elucidated on the basis of extensive spectroscopic analysis, including 1D NMR, 2D NMR, and HR-ESI-MS. ß 2013 Xian-Li Zhou and Jie Weng. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Orchidaceae Liparis nervosa Benzamide derivatives Nervogenic acid derivatives
2. Experimental
(1 L) at 50 8C and adjusted to pH 2.8 with 10% HCl, and extracted with CHCl3 and EtOAc to obtain the CHCl3 extract (82 g) and the EtOAc extract (25 g), respectively. The pH of the aqueous layer was adjusted to 9.4 with aqueous ammonia solution and extracted with EtOAc and n-butanol to afford the crude alkaline EtOAc extract (10 g) and the n-butanol extract (50 g), respectively. The n-butanol extract (50 g) was subjected to a column using D 101 resin and eluted with H2O, 30%, 60% and 95% EtOH, to afford four fractions (I, II, III, and IV). Fraction I (6.5 g) was subjected to RP-18 silica gel column, eluted in a step gradient manner with MeOH–H2O (1:50– 1:0) to afford fractions (A–F) based on TLC analysis. Fraction A (400 mg) was subjected to silica gel column, eluted with CHCl3:CH3OH:EtOAc (12:1:1) to yield compound 1 (50 mg), 2 (42 mg), 3 (23 mg), 4 (10 mg) and 5 (12 mg), respectively.
2.1. Plant material
2.3. Acid hydrolysis of 1–5
L. nervosa was collected in Chongqing, China in July 2007. The plant was identified by Professor Liangke Song in the School of Life Science and Engineering, Southwest Jiaotong University, Sichuan, China, where a voucher specimen is deposited (No. Z36150701).
Compound 1 (2 mg) was heated in 1 mol/L HCl–dioxane (1:1, 4 mL) at 80 8C for 4 h. After cooling, the solution was diluted with H2O (3 mL), neutralized with 1 mol/L NaOH and then extracted with CHCl3 (3 3 mL). The aqueous layer was concentrated under a stream of nitrogen. The residue was then dissolved in anhydrous pyridine (0.1 mL), followed by the addition of 0.1 mol/L L-cysteine methyl ester hydrochloride (0.2 mL). The resulting solution was stirred at 60 8C for 1 h, followed by the addition of trimethysilylation reagent hexamethyldisilazane–trimethylchlorosilane–pyridine (HMDS–TMCS–pyridine, 3:1:9). It was then stirred at 60 8C for an additional 30 min. The thiazolidine derivatives were analyzed by GC for sugar identification. GC analyses were performed using a Hewlett Packard GC6890 instrument on an
1. Introduction Liparis nervosa (Thunb. ex A. Murray) Lindl., a member of the Orchidaceae family, is an herbaceous plant widely distributed in China. It has been used in folk medicine for detoxification and hemostatic function [1]. Previous chemical investigations of L. nervosa led to the isolation of a series of nervogenic acid derivatives and pyrrolizidine alkaloids [2–5]. Our continued studies on the whole plant extract of L. nervosa have resulted in the isolation and structure determination of five new nervogenic acid derivatives (1–5) (Fig. 1).
2.2. Extraction and isolation The shade-dried L. nervosa (5.0 kg) was extracted with 95% EtOH (3 20 L). The EtOH extract (300 g) was suspended in H2O
* Corresponding authors. E-mail addresses: [email protected] (X.-L. Zhou), [email protected] (J. Weng).
1001-8417/$ – see front matter ß 2013 Xian-Li Zhou and Jie Weng. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. http://dx.doi.org/10.1016/j.cclet.2013.04.043
[(Fig._1)TD$IG]
S. Huang et al. / Chinese Chemical Letters 24 (2013) 734–736
O
b-D-glucopyranosyl units by gas chromatography of the hydro-
7 CR1
lyzed product and by the coupling constant of their anomeric protons Glc H-100 (d 4.74, d, J = 7.2 Hz) and Glc H-1000 (d 4.46, d, J = 7.8 Hz). In the HMBC experiment, long-range correlations were observed from Glc H-100 with C-4 (d 157.5) and Glc H-1000 with Glc C-400 (d 81.0). The linkage and the anomeric configuration of the sugar units agreed with the cellobiose unit of the nervosine III [5]. These data supported the structure of compound 1 as 3,5-bis(3methyl-2-butenyl)-4-O-[b-D-glucopyranosyl-(1!4)-b-D-glucopyranosyl] benzoic acid. Compound 2 was isolated as an amorphous solid. Its molecular formula was deduced as C28H40O12 by HR-ESI-MS at m/z 591.2417 [M+Na]+ (calcd. for C28H40O12Na: 591.2417). It was found to have a similar structure to compound 1 by comparison of their NMR data, the main difference observed was that a b-D-glucopyranoside unit was replaced by a a-L-arabinopyranose unit. It was confirmed by gas chromatography of the hydrolyzed product and by the coupling constant of their anomeric protons Ara H-100 (d 4.73, d, J = 7.6 Hz) and Glc H-1000 (d 4.77, d, J = 7.6 Hz). In the HMBC experiment, long-range correlations were observed from Ara H-100 with C-4 (d 158.1), Glc H-1000 with Ara C-200 (d 80.5). Moreover, the linkage of the sugar units was as same as in nervosine IV [5]. Thus, the structure of compound 2 was determined as 3,5-bis(3-methyl2-butenyl)-4-O-[b-D-glucopyranosyl-(1!2)-a-L-arabinopyranosyl] benzoic acid. Compound 3 was isolated as a white, amorphous solid and showed the molecular formula is C29H43NO12 by analysis of its HRESI-MS spectrum (m/z 620.2667 [M+Na]+, calcd. for C29H43NO12Na: 620.2683). The NMR spectroscopic data (Tables 1 and 2) of 3 were comparable to those of 1 and 2, indicating that 3 was also a nervogenic acid derivative. Comparison of the 13C NMR data (Table 2) of aglycon moiety of 3 with those of 3,5-bis(3methyl-2-butenyl)-4-O-(b-D-glucopyranosyl) benzamide [4], suggested that they possessed the same aglycone but differed in the sugar part. The sugar residues were identified as two Dglucopyranosides by gas chromatography. By analysis of the HMQC, HMBC and COSY data, compound 3 was determined to be 3,5-bis(3-methyl-2-butenyl)-4-O-[b-D-glucopyranosyl-(1!2)-bD-glucopyranosyl] benzamide.
R1
1 4'
3'
2'
3
1'
735
5'
5
6'
7'
OR2
8'
9'
10'
R2
1 OH
Glc ←1Glc
2 OH
Ara2←1Glc
3 NH2
Glc2←1Glc
4 NH2 5 NH2
4
Ara2←1Glc Glc4←1Glc
Fig. 1. Structures of compounds 1–5.
Agilent HP-5 column (0.25 mm, 30 m, i.d., 0.25 mm). Temperatures of both the injector and detector were 200 8C. A temperature gradient system was used for the oven, starting at 60 8C for 2 min and increasing up to 260 8C at a rate of 15 8C/min. D-Glucose and Larabinose were confirmed by comparison with the retention time of the authentic standards at 11.81 min and 9.04 min, respectively. The same analysis was carried out on compounds 2–5 [4]. 3. Results and discussion Compound 1 was isolated as a white, amorphous solid. Its molecular formula was determined as C29H42O13 by analysis of its HR-ESI-MS spectrum (m/z 599.2704 [M+H]+, calcd. for C29H43O13: 599.2706). The 1H NMR spectrum (Table 1) displayed a singlet at d 7.68 (s, 2H), indicating the presence of two aromatic protons. In addition, signals due to two prenyl groups were present, namely, d 5.31 (t, 2H, J = 7.2 Hz), d 3.59 (d, 4H, J = 7.2 Hz), and two singlets at d 1.75 (s, 6H) and d 1.77 (s, 6H), in total indicating 12 protons [4]. HMBC correlations were also observed from H-2 and H-6 (d 7.68) with the carbon resonance at dC 170.5 (C-7), indicating that the carboxyl group was attached to C-1. Comparison of the NMR data (Tables 1 and 2) of 1 with those published in literature [4], suggested that the aglycone moiety of 1 is 4-hydroxy-3,5-bis-(3methyl-2-butenyl)-benzoic acid, previously described as nervogenic acid unit [4]. The sugar residues were identified as two
Table 1 1 H NMR dataa of compounds 1–5. H
1b
2/6 10 /60 20 /70 40 /90 50 /100
7.68 3.59 5.31 1.75 1.77 Glc 4.74 3.59 3.60 3.68 3.29
100 200 300 400 500 600
10 00 20 00 30 00 40 00 50 00 60 00 a 1 b c
s d (7.2) t (7.2) s s d (7.2) m m m m
a 3.78 dd (2.0, 12.0) b 3.83 dd (6.4, 12.0) Glc 4.46 d (7.8) 3.26 m 3.35 m 3.31 m 3.36 m a 3.67 dd (5.6, 12.0) b 3.90 dd (2.0, 12.0)
H NMR (400 MHz, d, J in Hz). Data in CD3OD. Data in DMSO-d6.
2b
3b
7.66 s 3.54 d (7.2) 5.32 t (7.2) 1.73 s 1.76 s Ara 4.73 (7.6) 4.16 m 3.84 m 3.86 m a 3.40 overlapped b 3.90 dd (2.8, 8.4) –
7.67 3.45 5.21 1.67 1.71 Glc 4.81 3.82 3.43 3.40 3.46
Glc 4.77 d (7.6) 3.35 m 3.40 m 3.35 m 3.30 m a 3.67 dd (5.0, 11.3) b 3.80 dd (2.2, 11.3)
4c s d (7.2) t (7.2) s s overlapped m m m m
a 3.77 dd (7.0, 12.0) b 3.82 dd (2.2, 12.0) Glc 4.80 overlapped 3.31 m 3.14 m 3.30 m 3.43 m a 3.67 dd (5.2, 12.0) b 3.62 brd (12.0)
7.48 3.55 5.29 1.69 1.69 Ara 4.72 4.01 3.74 3.64 3.33 3.81 –
5c s d (7.6) t (7.6) s s d (5.2) m m m brd (11.6) dd (2.0, 11.6)
Glc 4.48 d (7.6) 3.02 m 3.17 m 3.10 m 3.40 m a 3.60 dd (5.2, 11.6) b 3.39 overlapped
7.48 3.54 5.27 1.70 1.70 Glc 4.65 4.04 3.72 3.54 3.21
s d (7.6) t (7.6) s s d (6.4) m m m m
a 3.47 overlapped b 3.72 brd (11.2) Glc 4.31 d (7.2) 3.00 m 3.16 m 3.07 m 3.30 m a 3.53 overlapped b 3.66 brd (11.8)
S. Huang et al. / Chinese Chemical Letters 24 (2013) 734–736
736 Table 2 13 C NMR dataa of compounds 1–5. C
1b
1c
2b
3b
4c
5c
1 2/6 3/5 4 7 10 /60 20 /70 30 /80 40 /90 50 /100
128.6 s 130.3 d 136.9 s 157.5 s 170.5 s 29.6 t 124.0 d 133.9 s 18.1 q 26.0 q Glc-I 105.7 d 76.3 d 75.4 d 81.0 d 76.7 d 62.0 t Glc-II 104.6 d 74.9 d 77.9 d 71.4 d 78.3 d 62.4 t
126.9 128.8 135.7 156.4 167.4 28.2 123.1 132.4 18.1 25.8 Glc-I 104.5 d 74.9 d 74.0 d 80.5 d 75.1 d 60.7 t Glc-II 103.3 d 73.5 d 76.7 d 70.3 d 77.1 d 61.3 t
127.9 s 130.3 d 137.0 s 158.1 s 169.9 s 29.6 t 123.8 d 134.0 s 18.1 q 26.0 q Ara 105.8 d 80.5 d 73.9 d 69.1 d 66.7 t – Glc 104.9 d 75.7 d 78.0 d 71.5 d 78.0 d 62.7 t
131.3 s 128.3 d 137.3 s 156.6 s 172.5 s 29.8 t 124.3 d 133.4 s 18.3 q 26.0 q Glc-I 104.3 d 82.9 d 78.1 d 71.4 d 78.4 d 62.7 t Glc-II 105.4 d 76.0 d 78.1 d 71.7 d 78.1 d 63.0 t
130.5 s 127.1 d 135.1 s 155.5 s 168.2 s 28.7 t 123.3 d 132.0 s 18.2 q 25.9 q Ara 104.4 d 79.5 d 71.4 d 66.4 d 64.6 t – Glc 103.1 d 74.4 d 76.8 d 70.2 d 77.1 d 61.2 t
130.7 s 127.1 d 135.3 s 154.9 s 168.1 s 28.4 t 123.5 d 131.8 s 18.1 q 25.9 q Glc-I 104.5 d 74.9 d 74.0 d 80.5 d 75.0 d 60.7 t Glc-II 103.3 d 73.5 d 76.7 d 70.3 d 77.0 d 61.2 t
100 200 300 400 500 600 100 0 200 0 300 0 400 0 500 0 600 0
the HMBC experiment. Thus the structure of compound 4 was determined as 3,5-bis(3-methyl-2-butenyl)-4-O-[b-D-glucopyranosyl-(1!2)-a-L-arabinopyranosyl] benzamide. The molecular formula of 5 was established as C29H43NO12 on the basis of the HR-ESI-MS. Comparison of the NMR data of compounds 5 and 4 (Tables 1 and 2) indicated that 5 possessed the same aglycone moiety. Complete assignment of the glycosidic protons and carbons along with an acid hydrolysis indicated the presence of two b-D-glucopyranosyl units, which has a (1!4) linkage by comparison of the NMR data with those of compound 1. By analysis of the HMQC, HMBC and COSY data, 5 was elucidated as 3,5-bis(3-methyl-2-butenyl)-4-O-[b-D-glucopyranosyl-(1!4)-bD-glucopyranosyl] benzamide. Acknowledgments This research work was financially supported by the National Natural Science Foundation of China (Nos. 21142004, 31171695), the New Century Talents Scheme of the Ministry of Education (No. NCET-08-0820), Science and Technology Support Programs of Sichuan Province (No. 2013SZ0083), and the Fundamental Research Funds for Central Universities (Nos. SWJTU2010ZT09, SWJTU12CX048).
a 13 b c
C NMR (100 MHz, d). Data in CD3OD. Data in DMSO-d6.
Compound 4 was isolated as an amorphous solid. Its molecular formula was deduced as C28H41NO11 by HR-ESI-MS at m/z 568.2744 [M+H]+ (calcd. for C28H42NO11: 568.2758). The HR-ESIMS and 13C NMR spectra (Table 2) revealed that compound 4 was also a benzamide derivative. After hydrolysis, sugars were identified as D-glucopyranoside and L-arabinopyranoside, in a ratio of 1:1, based on the GC analysis of their chiral derivatives. Their sequencing was identified as the same as in compound 2 by
References [1] Y.X. Hua, S.F. Liu, Z.Q. Yang, The Chinese Herbal, vol. 12, Shanghai Science and Technology Publishing House, Shanghai, 1999p. 997. [2] K. Nishikawa, Y. Hirata, Chemotaxonomical alkaloid studies. I. Structure of nervosine, Tetrahedron Lett. 27 (1967) 2591–2596. [3] K. Nishikawa, M. Miyamura, Y. Hirata, Chemotaxonomical alkaloid studies structures of Liparis alkaloids, Tetrahedron 25 (1969) 2723–2741. [4] S. Huang, X.L. Zhou, C.J. Wang, et al., New nervogenic acid derivatives from Liparis nervosa, Planta Med. 79 (2013) 281–287. [5] S. Huang, X.L. Zhou, C.J. Wang, et al., Pyrrolizidine alkaloids from Liparis nervosa with inhibitory activities against LPS-induced NO production in RAW264.7 macrophages, Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013. 03.009.