New Stemona alkaloids from the roots of Stemona tuberosa.

Letter - spectral assignment Received: 22 February 2014

Revised: 4 June 2014

Accepted: 6 June 2014

Published online in Wiley Online Library: 30 June 2014

(wileyonlinelibrary.com) DOI 10.1002/mrc.4099

New Stemona alkaloids from the roots of Stemona tuberosa Yong Yue,a,b An-Jun Deng,a,b Zhi-Hong Li,a,b Ai-Lin Liu,a,b Lin Ma,a Zhi-Hui Zhang,a,b Xiu-Li Wang,c Gu-Hua Dua,b and Hai-Lin Qina,b*

Introduction

Magn. Reson. Chem. 2014, 52, 719–728

Results and discussion The dried root of S. tuberosa was extracted with ethanol. The extract was further purified by multiple procedures to give compounds 1-5 (Fig. 1). All the five new compounds were elucidated to be Stemona alkaloids sharing 22 carbon atoms by their molecular formulae obtained from the HRESIMS of positive ion mode (refer to experimental section). The category of Stemona alkaloids was also determined by their common features from NMR spectroscopic data, with identical 22 carbon signals being exhibited in each of the five 13C NMR spectra, and with one primary methyl group and two secondary methyl groups being detected in higher field region and two unambiguous methineoxy groups being indicated in the relatively lower field region, respectively, in each of the five 1H NMR spectra (Tables 1–5). All of these characteristics were confirmed by HSQC and DEPT experiments. The three methyl groups belong to Me-17, 15, and 22 and the two

* Correspondence to: Hai-Lin Qin, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China. E-mail: [email protected] a State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China b Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China c High and New Technology Research Center of Henan Academy of Sciences, Zhengzhou 450002, China

Copyright © 2014 John Wiley & Sons, Ltd.

719

Stemona tuberosa Lour. (Stemonaceae) is one of the original plants of Stemonae radix that was documented in the Chinese Pharmacopoeia as ‘Baibu’. Just like the other two original plants, Stemona sessilifolia Miq. and Stemona japonica Miq., the roots of S. tuberosa have been widely used for thousands of years for treatment of respiratory illness, such as pertussis and tuberculosis, in China, Korea, Japan, and some other Asian countries.[1,2] Chemically, Stemona alkaloids are generally accepted as the most important constituents from Stemona species, and it has been reported that some 150 Stemona alkaloids have been isolated to date. Stemona alkaloids are one class of the most complicated alkaloids, with multiple chiral carbons existing in their structures in general. The discovery and structure elucidation of new Stemona alkaloids have always been one of the most interesting works in natural medicinal chemistry. Stemona alkaloids were structurally classified by Pilli et al. into eight groups, six of which share the pyrrolo[1,2-a]azepine core characteristic of the majority of Stemona alkaloids, the seventh group displays the pyrido[1,2-a] azepine nucleus, and the last miscellaneous group is formed by those alkaloids lacking these two basic structural motifs, or are until the recent publication of Pilli’s classification the sole representative of a new group.[3] The former six groups were further characterized according to the sites of connection between the basic pyrrolo[1,2-a]azepine core and the side chain, and the miscellaneous group comprising a relatively small number of Stemona alkaloids was reported to be mainly produced from the common Stemona alkaloids via oxidation or rearrangement processes.[3–10] Also, based on structural considerations and their various distribution in different species, H. Greger classified Stemona alkaloids into three skeletal types: the stichoneurine-type (tuberostemonine type), protostemonine-type, and croomine-type alkaloids. Among which, the stichoneurine-type was characterized by a carbon chain attached to C-9 of Stemona alkaloids and with an ethyl group (C-16–C-17) attached to C-10 of the carbon chain being common.[11] In our previous paper, three moderately acetylcholinesterase (AChE)-inhibiting Stemona alkaloids sharing a tetracyclic decahydro-1H-furo[2′,3′:4,5]cyclopenta[1,2-b]pyrrolo [1,2-a]azepine nucleus were described from the roots of S. sessilifolia, which was exclusive to all known groups then according to the early Pilli’s classification system published in 2000.[12] Some new Stemona alkaloids were also reported by our group in recent years from S. sessilifolia and S. tuberosa,[2,13–15] respectively. In our ongoing search for AChE-inhibiting natural compounds, five new Stemona alkaloids (1–5) of the miscellaneous group according to

Pilli’s classification,[3] or tuberostemonine group according to Greger’s classification, were isolated from the EtOH extract of the roots of S. tuberosa. This paper mainly deals with the isolation and structural elucidation of these new compounds. Some of the isolated compounds were tested for the AChE-inhibiting and butyrylcholinesterase (BuChE)-inhibiting activities in vitro. As a result, these compounds showed nearly no BuChE-inhibiting effects at the concentration of 5 μg ml1, with inhibitory ratios (IRs) ranging from 21.2 to 25.8% in contrast with 68.67% of positive control, tetraisopropylpyrophosphoramide (iso-OMPA).

Y. Yue et al.

Figure 1. The structures of compounds 1–5.

Table 1. NMR spectroscopic data (in DMSO-d6, TMS) for stemonatuberone A (1) Position 1 2α

1

δ H (J in Hz)

δ

13

C

DEPT

Coupling observed in the HMBC experiment (H → C)

NOE correlations observed in the ROESY experiment

210.5 39.8

C CH2

C-1, C-3, C-18

H-3, H-2β, H-19α

68.3

CH

C-1, C-3, C-18 C-1, C-5, C-9a, C-18, C-19

3.05 br d (14.0) 3.35 m

52.9

CH2

H-2α H-2α, H-2β, H-5ex, H-5en (weak), H-18, H-19α, H-19β H-3, H-5en, H-7a

6a,b 7a 7b 8a,b 9 9a 10

1.50 ov 1.70 m 1.50 ov 1.50 ov 2.52 m

27.7 23.8a

CH2 CH2

23.2a 49.3 177.0b 36.5

CH2 CH C CH

11

4.80 dd (9.5 and 6.5) 3.65 dd (6.5) 2.70 dq (6.5 and 7.0)

80.3

CH

C-1, C-9, C-10, C-12, C-14, C-16

58.4

CH

39.0 ov

CH

2β 3 5ex 5en

12 13 14 15 16 17 18 19α 19β 20 21 22

2.38 dd (10.5 and 5.5) 3.50 ov 3.50 ov

2.15 m

1.14 d (7.0) 1.47-1.56 ov 0.80 t (7.5) 5.04 m 1.42 m 2.27 ddd (14.0, 8.5, and 5.5) 2.75 m 1.09 d (7.0)

H-3 (weak), H-5ex, H-10, H-7a (weak)

C-9

177.2b 14.5c 19.6 7.4 77.9 33.6

C CH3 CH2 CH3 CH CH2

35.0 178.6 14.4c

CH C CH3

C-7, C-8, C-10, C-11

H-10, H-11, Me-17

C-9, C-17

C-1, C-10, C-11, C-13, C-14, C-15

H-5en, H-9, H-11, H-13 (weak), H2-16, Me-17 H-9, H-10, H-12, Me-15, Me-17 H-11, H-13, Me-15

C-1, C-11, C-12, C-14, C-15

H-10 (weak), H-12, Me-15

C-12, C-13, C-14

H-11, H-12, H-13

C-10, C-16 C-3 C-3, C-18, C-20, C-22 C-20, C-21

H-9, H-11, H-16 H-2β, H-3, H-19α, H-19β, H-20 H-2α, H-2β, H-3, H-18, H-19β, H-20 H-3, H-18, H-19α, H-20

C-19, C-21, C-22

H-18, H-19β, Me-22

C-19, C-20, C-21

H-19α, H-19β, H-20

ov, signal overlapping; en, endo; ex, exo. Signals with the same superscript letters in the same column can be exchanged.

a, b , c

720

methineoxy groups to CH-11 and 18 of the skeleton of most tuberostemonine-type of Stemona alkaloids, respectively. Compound 1 was isolated as a white amorphous powder. The molecular formula was determined by the aforementioned HRESIMS measurement to be C22H31NO6, indicating eight unsaturation degrees. The IR absorptions at 1767 and

wileyonlinelibrary.com/journal/mrc

1704 cm1 indicated the presence of γ-lactone moities. The DEPT spectrum classified the 22 carbons into 3 methyl groups, 7 methylene groups, 8 methine groups, and 4 quaternary carbons. Detailed analysis of 1D and 2D NMR spectroscopic data of 1 and a comparison of them with those of neotuberostemonone, a known Stemona alkaloid of tuberostemonine type isolated from


Magn. Reson. Chem. 2014, 52, 719–728

Structure elucidations of new Stemona alkaloids Table 2. NMR spectroscopic data (in DMSO-d6, TMS) for stemonatuberone B (2) Position 1 2α 2β 3 5ex 5en 6a,b 7a 7b 8a 8b 9 9a 10 11 12 13 14 15 16a 16b 17 18 19α 19β 20 21 22

1

δ H (J in Hz)

2.97 dd (12.0 and 12.0) 2.12 dd (12.0 and 5.0) 5.02 ddd (12.0, 10.0, and 5.0) 3.36 ddd (15.0, 4.0, and 4.0) 3.84 m 1.75 ov 1.87 m 1.35 m 1.67 ov 1.50 m 2.75 m 2.15 m 4.92 dd (10.5 and 5.5) 3.67 dd (5.5 and 3.5) 2.65 qd (7.5 and 3.5) 1.26 d (7.5) 1.67 ov 1.30 m 0.89 d (7.0) 4.66 ddd (10.0, 10.0, and 5.5) 1.62 m 2.35 m 2.80 m 1.13 d (7.0)

δ

13

C

DEPT



H-2β, H-3, H-12, H-18 H-2α, H-3, H-18, H-19α, H-19β H-2α, H-2β, H-5ex, H-18, H-19α

207.7 42.7

C CH2

56.3

CH

C-1, C-3, C-12, C-18 C-1, C-3, C-12 C-1, C-2, C-5, C-9a, C-18

38.6

CH2

C-3, C-6, C-7, C-9a

H-3, H-5en, H-18

C-3

H-5ex, H-11, H-12

25.2 20.9

CH2 CH2

C-9 C-9

H2-6, H-7b, H-8a H-7a

18.0

CH2

50.2 176.9a 46.0 80.6 56.6 40.3 177.0a 15.1 23.3

CH C CH CH CH CH C CH3 CH2

C-7, C-9, C-9a C-9a, C-11

H-7a, H-7b, H-8a,H- 9 H-10, Me-17

C-11 C-1, C-10, C-12, C-14, C-16 C-1, C-13, C-14, C-15 C-1, C-11, C-12, C-14, C-15

H-9, H-11, Me-17 H-5en, H-10, H-12, Me-15, H-16 H-2α, H-5en, H-11, H-13, Me-15 H-12, Me-15

C-12, C-13, C-14

H-11, H-12, H-13

11.8 74.7

CH3 CH

33.9

CH2

35.0 178.5 14.5

CH C CH3

C-10, C-11, C-17 C-10, C-16 C-3, C-19 C-20, C-21 C-3, C-18, C-20, C-22 C-19, C-22

H-9, H-10, H-16 H-2α, H-3, H-5ex, H-19α (weak), H-19β, H-20 H-2β, H-3, H-18, H-19β, H-20, Me-22 H-2β, H-3, H-18, H-19α, H-20 H-18, H-19β, Me-22

C-19, C-20, C-21

H-19α, H-20

ov, signal overlapping; en, endo; ex, exo. Signals can be exchanged.

a

Magn. Reson. Chem. 2014, 52, 719–728

18/H-20 confirmed the cis relationship between H-18 and H-20 in 1 (Fig. 2). Other detailed key NOE correlations were given in Table 1. Thus, the structure of compound 1 was established as (rel)-(3S*,3aS*,6S*,12R*,13R*,13aS*)-13-ethyl-3-methyl-6-((2S*,4S*)-4methyl-5-oxotetrahydrofuran-2-yl)decahydro-2H-7,12-methanofuro [3,2-e][1]azacyclododecine-2,4,14(8H)-trione. Complete assignments of 1H and 13C NMR signals of 1 were performed by detailed 1D and 2D NMR experiments (mainly including 1H NMR, 13C NMR, and DEPT NMR and 1H–1H COSY, HSQC, HMBC, and ROESY experiments, Table 1), and the trivial name of stemonatuberone A was given to it. Compound 2 was isolated as colorless crystals and was determined by HRESIMS measurement to possess the molecular formula C22H31NO6. The IR absorptions at 1782 and 1709 cm1 indicated the presence of γ-lactone moieties. The DEPT spectrum also classified the 22 carbons into 3 methyl groups, 7 methylene groups, 8 methine groups, and 4 quaternary carbons. A detailed comparison of NMR data between compounds 2 and 1, as well as with those of neotuberostemonone,[16] indicated that they have not only the very identical categories of functional groups with one another described by the previously described DEPT NMR spectrum but also noticeable differences in terms of chemical shifts (Tables 1 and 2). After the 1H NMR signals were



721

Stemona mairei,[16] revealed not only the very similar spectroscopic features between 1 and neotuberostemonone, mainly their identical categories of functional groups, but also noticeable differences in terms of chemical shifts. The HMBC experiment confirmed that the gross structure of compound 1 was exactly commensurate with that of neotuberostemonone, with the following key long range 1H–13C correlations being evident: from Me-17 to C-10 and C-16; from H-11 to C-1, 9, 10, 12, 14, and C16; from Me-15 to C-12, 13, and C-14; from H-22 to C-19, 20, and C-21; from H-12 to C-1, 10, 11, 13, 14, and C-15; from H-13 to C-1, 11, 12, 14, and C-15; from H-3 to C-1, 5, 9a, 18, and C-19; from H-2α to C-1, 3, and C-18; and from H-2β to C-1, 3, and C18 (Table 1). Considering this information, it was affirmed that the stereoconfiguration of 1 was different from that of neotuberostemonone. The relative configuration of 1 was thus determined by a ROESY experiment coupled with the biogenetic consideration. Starting from the common α-orientation of H-3 in Stemona alkaloids, the NOE correlations of H-3/H-5ex, H-3/H-5en (weak), and H-5en/H-10 in 1 indicated that H-10 was α-oriented, confirming the β-orientation of H-9. The NOE correlation between H-10 and H-13 indicated the α-orientation of H-13, meaning that H-11, 12, and Me-15 were β-oriented. The NOE correlation of H-

Y. Yue et al. Table 3. NMR spectroscopic Data (in DMSO-d6, TMS) for stemonatuberone C (3) Position 1 2a 2b 3 5ex 5en 6 7 8a 8b 9 9a 10 11 12 13 14 15 16a 16b 17 18 19α 19β 20 21 22

1

δ H (J in Hz)

3.22 ov 2.54 ov 5.07 m 3.22 ov 3.01 dd (14.5 and 12.0) 1.45–1.65 m 1.45–1.65 m 1.84 m 1.32 m 2.70 m 2.32 br dd (11.0 and 11.0) 4.63 dd (11.0 and 5.0) 3.37 d (5.0) 2.54 ov 1.27 d (7.5) 1.68 ov 1.44 ov 0.80 t (7.5) 4.65 m 1.56 m 2.42 m 2.76 m 1.13 d (7.0)

δ

13

C

DEPT

209.9 46.9

C CH2

55.6 42.3

CH CH2

23.7a 23.4 27.8a

CH2 CH2 CH2



C-2, C-5, C-9a, C-18, C-19

H-2b, H-3, H-18 H-2a, H-3, H-18 H-2a, H-2b, H-5ex, H-12, H-18, H-19α H-3, H-5en H-5ex, H-10

48.2 178.6 36.0

CH C CH

C-7, C-9a

H-10, H-11, Me-17

C-9a, C-17

H-5en, H-9, H-11

80.6 51.9 41.5 177.3 15.3 19.3

CH CH CH C CH3 CH2

C-1, C-9, C-10, c-16 C-1, C-13, C-14, C-15

H-9, H-10, H-12, Me-15 (weak), Me-17 H-3, H-11, H-13, Me-15 H-12, Me-15

C-12, C-13, C-14 C-9, C-10, C-17

H-11 (weak), H-12, H-13 H-10, Me-17

6.5 77.3 33.8

CH3 CH CH2

34.3 178.6 15.0

CH C CH3

C-10, C-16 C-2, C-3 C-3, C-18, C-20, C-22 C-21, C-22 C-19, C-21, C-22

H-9, H-11, H-16 H-2a, H-2b, H-3, H-19α, H-20, H-19β H-3, H-18, H-19β, H-20, Me-22 H-18, H-19α, H-20 H-18, H-19α, H-19β, Me-22

C-19, C-20, C-21

H-19α, H-19β, H-20


a

722

correlated to their corresponding carbon signals by the HSQC spectrum, the very identical gross structures between 2 and 1 were confirmed by the HMBC experiment that showed the following long-range 1H–13C correlations in 2: from Me-17 to C-10 and C-16; from H-11 to C-1, 10, 12, 14, and C-16; from Me-15 to C-12, 13, and C-14; from Me-22 to C-19, 20, and C-21; from H-12 to C-1, 13, 14, and C-15; from H-13 to C-1, 11, 12, 14, and C-15; from H-3 to C-1, 2, 5, 9a, and C-18; from H-2α to C-1, 3, 12, and C-18; from H-2β to C-1, 3, and C-12; from H-5en to C-3; and from H-5ex to C-3, 6, 7, and C-9a (Table 2). Considering the previous information, it was affirmed that the stereoconfiguration of compound 2 was different from those of 1 and neotuberostemonone. As in the case of compound 1, the relative configuration of 2 was also elucidated by the ROESY experiment and starting from the common α-orientation of H-3 in Stemona alkaloids. The NOE correlations of H-3/H-5ex, H-5en/H-11, and H-5en/H-12 indicated the α-orientation of H-11 and H-12, meaning that H-9 was β-oriented. The NOE correlations of H-9/Me-17 and H-11/Me-15 revealed the α-orientation of H-10 and Me-15. Similarly, the NOE correlation of H-18/H-20 confirmed the cis relationship between H-18 and H-20 in 2 (Fig. 3). Thus, the structure of compound 2 was established as (rel)-(3R*,3aR*,6S*,12R*,13R*,13aR*)-13-ethyl-3-methyl-6-((2S*,4S*)4-methyl-5-oxotetrahydrofuran-2-yl)decahydro-2H-7,12-methanofuro[3,2-e][1]azacyclododecine-2,4,14(8H)-trione. Complete assignments of 1H and 13C NMR signals of 2 were also performed by


detailed 1D and 2D NMR experiments (mainly including 1H NMR, 13C NMR, and DEPT NMR and 1H–1H COSY, HSQC, HMBC, and ROESY experiments, Table 2), and the trivial name of stemonatuberone B was given to 2. Compound 3 was isolated as a white amorphous powder. It was affirmed by HRESIMS measurement to have the same molecular formula of C22H31NO6 as compounds 1 and 2. The IR absorptions at 1773 and 1700 cm1 also indicated the presence of γlactone moieties in 3. The DEPT spectrum also classified the 22 carbons into the same 3 methyl groups, 7 methylene groups, 8 methine groups, and 4 quaternary carbons as compounds 1 and 2. Comparison of the 1H and 13C NMR spectra indicated that 3 was different in chemical shifts from those of 1 and 2, as well as neotuberostemonone, albeit the previously mentioned identical categories of functional groups among them, which suggested that the stereoconfiguration of 3 was different from those of the other three compounds. After the 1H NMR signals were correlated to their corresponding carbon signals by the HSQC spectrum, the HMBC experiment confirmed that compound 3 has identical constitution with compounds 1, 2, and neotuberostemonone, which showed the following long-range 1H–13C correlations in 3: from H-10 to C-9a and C-17; from H-11 to C-1, 9, 10, and C-16; from H-12 to C-1, 13, 14, and C-15; from H-16b to C-9, 10, and C-17; from Me-15 to C-12, 13, and C-14; from Me-17 to C-10 and C-16; from H-18 to C-2 and C-3; from Me-22 to C-19,


Magn. Reson. Chem. 2014, 52, 719–728

Structure elucidations of new Stemona alkaloids Table 4. NMR spectroscopic data (in DMSO-d6, TMS) for stemonatuberonol A (4) Position 1 2α 2β 3 5en 5ex 6a 6b 7a 7b 8α 8β 9 9a 10 11 12 13 14 15 16a,b 17 18 19α 19β 20 21 22 OH

1

δ H (J in Hz)

2.82 dd (12.0 and 7.5) 2.48 ov 3.55 ddd (10.0, 7.5, and 4.0) 3.75 m 3.21 dd (14.5 and 5.5) 1.96 m 1.41 ov 1.89 m 1.41 ov 1.78 br d (14.0) 1.20 m 2.64 br d (12.0)

4.91 d (7.0) 3.76 dd (11.5 and 7.0) 3.06 dq (11.5 and 7.5) 0.95 d (7.5) 1.49 q (7.5) 0.81 t (7.5) 5.29 ddd (10.0, 10.0, and 5.5) 1.40 m 2.48 ov 2.84 m 1.12 d (7.0) 4.53 s

δ

13

C

DEPT


208.0 41.0

C CH2

C-1, C-3, C-18

66.1

CH

C-1, C-3, C-18 C-1, C-9a, C-18, C-19

51.4

CH2

28.7a

CH2

28.1a

CH2

22.5

CH2

49.9

CH

C-7, C-8, C-9a, C-10

180.9 76.4 89.9 51.2

C C CH CH

C-1, C-10, C-12, C-14, C-16 C-1, C-10, C-11, C-13, C-14, C-15

36.8 177.5 12.3 26.7 8.4 78.3

CH C CH3 CH2 CH3 CH

35.1

CH2

35.2 178.7 14.6

CH C CH3

C-7 C-3, C-7, C-9a

C-1, C-11, C-12, C-14, C-15


H-2β, H-3, H-5en, H-12, Me-15, H-19β H-2α, H-3, Me-15, H-19α H-2α, H-2β, H-5ex, H-18, H-19α H-5ex, H-6b, H-9 H-3, H-5en, H-6b, H-5ex, H-5en, H-6b, H-7a H-7a H-6a, H-6b, H-8α, H-8β H-8β, H-9, Me-17 H-7a, H-8α, H-9 H-5en, H-8α, H-8β, H-12, H2-16, Me-17

OH-10, H-12, Me-15 H-2α, H-9, H-11, H-13, Me-15, Me-17 H-12, Me-15, Me-17

C-12, C-13, C-14 C-9, C-10, C-11, C-17 C-10, C-16 C-3

H-2α, H-2β, H-11, H-13

C-3, C-18, C-20, C-22 C-3, C-18, C-20, C-21 C-18, C-19, C-21, C-22

H-2α, H-3, H-19β H-3, H-18, H-19α, H-20 H-18 (weak) , H-19β, Me-22

C-19, C-20, C-21 C-9, C-10, C-11, C-16, C-17

H-20 H-8α, H-8β, H-11

H-13, H-8α, H-9, H-12 H-2β, H-3, H-19β, H-20 (weak)


a

Magn. Reson. Chem. 2014, 52, 719–728

described compounds 1–3. The IR absorptions at 3501, 1772, and 1709 cm1 indicated the presence of a hydroxyl group and γ-lactone moieties. Except for the aforementioned general 1H NMR features of Stemona alkaloids exhibited in 4, one deshielded methine group at δH 3.55 (1H; ddd; J = 10.0, 7.5, and 4.0; H-3) and one labile proton at δH 4.53 (1H, s) were also exhibited in the 1H NMR spectrum of 4. The labile proton was confirmed by the HSQC experiment that showed that no 13C NMR signals correlated with the 1H NMR signal at δH 4.53. The DEPT NMR spectrum classified the 22 carbons into 3 methyl groups, 7 methylene groups, 7 methine groups, and 5 quaternary carbons. Comparison of the 1H and 13C NMR spectroscopic data of 4 with those of 1 (also including 2 and 3) indicated that they were very similar. The main difference was that the characteristic methine signal at δH 2.15 (1H, m, H-10) and δC 36.5 (d, C-10) in 1 was substituted by the similarly characteristic signal of one bearing an oxygen quaternary carbon at δC 76.4 (s) in 4. After the 1H NMR signals were correlated to their corresponding carbon signals by the HSQC experiment, the HMBC experiment confirmed the hydroxyl group



723

20, and C-21; and from H-3 to C-2, 5, 9a, 18, and C-19. Also, the relative configuration of 3 was resolved by the ROESY experiment. Starting with the common α-orientation of H-3 in Stemona alkaloids, the NOE correlations of H-3/H-12 and H-5en/H-10 indicated the α-orientation of H-12 and H-10, as well as the β-orientation of H-9. The observation that no NOE correlation was observed between H-11 and H-13 but NOE correlations of H-9/ H-11 and H-11/Me-15 (weak) were observed revealed and confirmed the β-orientation of H-11 and Me-15. Similarly, the NOE correlation of H-18/H-20 confirmed the cis relationship between H-18 and H-20 in 3 (Fig. 4). Other detailed NOE correlations were given in Table 3. Thus, the structure of compound 3 was established as (rel)-(3S*,3aR*,6S*,12R*,13R*,13aS*)-13-ethyl-3methyl-6-((2S*,4S*)-4-methyl-5-oxotetrahydrofuran-2-yl)decahydro2H-7,12-methanofuro[3,2-e][1]azacyclododecine-2,4,14(8H)-trione and was given the trivial name stemonatuberone C. Compound 4 was isolated as a white amorphous powder. The molecular formula was determined by HRESIMS measurement to be C22H31NO7, i.e. one oxygen atom more than the previously

Y. Yue et al. Table 5. NMR spectroscopic data (in DMSO-d6, TMS) for stemonatuberosine A (5) Position 1 2α 2β 3 5ex 5en 6a 6b 7a 7b 8a 8b 9 9a 10 11 12 13 14 15 16a 16b 17 18 19a 19b 20 21 22 OH

1

δ H (J in Hz)

1.70 dd (13.0 and 4.0) 1.99 dd (13.0 and 13.0) 3.58 ddd (13.0, 10.5, and 4.0) 3.25 dd (14.5 and 3.5) 3.44 m 1.59 m 1.38 m 1.75 m 1.17 ov 1.54 m 1.17 ov

2.31 m 4.56 dd (7.5 and 5.0) 2.34 br d (7.5) 2.57 br q (7.5) 1.19 d (7.2) 1.86 dqd (13.0, 7.5, and 2.5) 1.34 m 0.99 t (7.5) 4.63 ddd (10.5, 10.5, and 5.5) 1.41 m 2.45 m 2.78 m 1.12 d (7.0) 5.33 s

δ

13

C

DEPT


81.3 30.1

C CH2

58.5 47.4

CH CH2

32.5

CH2

26.0

CH2

C-6

28.1

CH2

C-6, C-7, C-9, C-9a, C-10

61.1 184.6 49.8 86.8 52.0 34.2 180.3 16.9 21.8

C C CH CH CH CH C CH3 CH2

13.2 76.5 34.8

CH3 CH CH2

34.3 178.7 14.6

CH C CH3

C-1, C-3, C-9 C-1, C-3, C-18 C-2, C-5, C-18 C-7, C-9a C-3, C-7


OH-1, H-2β, H-3, H-13, H-19α, H-19β H-2α, H-3, H-12 (weak) , H-18, Me-15 OH-1, H-2α, H-5ex, H-18, H-19a H-3, H-5en, H-6a, H-6b, H-18 H-5ex, H-6a H-5en H-5ex H-7b H-7a H-8b, H-16b H-8a

C-9, C-9a, C-11, C-16, C-17 C-1, C-9, C-14, C-16 C-11, C-13, C-14, C-15 C-1, C-11, C-12, C-14, C-15

H-11, H-16a, H-16b, Me-17 H-10, H-12, Me-15, H-16b, Me-17 H-11, H-13, Me-15, Me-17 OH-1, H-12, Me-15

C-12, C-13, C-14 C-10, C-11, C-17 C-10, C-11, C-17 C-10, C-16 C-3, C-19, C-20 C-3, C-18, C-20, C-22 C-20, C-21 C-19, C-21, C-22

H-2β, H-11, H-12, H-13 H-10, Me-17 H-8a, H-10, H-11, Me-17 H-10, H-11, H-12, H-16a, H-16b H-2, H-3, H-5ex, H-19a, H-19bβ, H-20 H-2α, H-3, H-19b H-2α, H-18, H-19a, H-20 H-18, H-19b, Me-22

C-19, C-20, C-21 C-1, C-2, C-9

H-20 H-2α, H-3, H-12 (weak), H-13

ov, signal overlapping; en, endo; ex, exo.

724

to be linked to C-10 with the following long-range 1H–13C correlations being obtained: from Me-17 to C-10 at δC 76.4 and C-16 at δC 26.7 and from δH 4.53 (OH-10) to C-9 at δC 49.9, 10 and 11 at δC 89.9, and 16 and C-17 at δC 8.4. Other key HMBC cross peaks confirming the constitution of 4 were shown in Table 4. The relative configuration of 4 was also determined by the ROESY experiment and starting with the common α-orientation of H-3 in Stemona alkaloids. The bold NOE correlations of H-3/H-5ex, H5en/H-9, H-9/H-12, H-9/Me-17, and H-12/Me-17 indicated the αorientation of H-9 and H-12, as well as the β-orientation of OH10. In addition, coupling with the examination of a molecular model, the observations that significant NOE correlations of OH10/H-11, OH-10/H-8α, OH-10/H-8β, Me-17/H-8α, and H-11/Me-15 were observed and that no NOE correlations were observed between Me-17 and H-11 and between Me-17 and H-8β revealed the β-orientation of H-11 and Me-15. Similarly, the NOE correlation of H-18/H-20 confirmed the cis relationship between H-18 and H-20 in 4 (Fig. 5). Other detailed NOE correlations were given in Table 4. Thus, the structure of compound 4 was established as (rel)-(3S*,3aR*,6S*,12S*,13R*,13aR*)-13-ethyl-13-hydroxy-3-methyl6-((2S*,4S*)-4-methyl-5-oxotetrahydrofuran-2-yl)decahydro-2H-7,12methanofuro[3,2-e][1]azacyclododecine-2,4,14(8H)-trione and was given the trivial name stemonatuberonol A.


Compound 5 was isolated as a white amorphous powder. It has a molecular formula of C22H31NO6 as determined by the positive HRESIMS. The IR absorptions at 3435, 1767, and 1704 cm1 indicated the presence of γ-lactone moieties and hydroxyl group. Except for the aforementioned common 1H NMR features of Stemona alkaloids in 5, one deshielded methine group at δH 3.58 (1H; ddd; J = 13.0, 10.5, and 4.0; H-3) and one labile proton at δH 5.33 (s) were also detected in the 1H NMR spectrum. The labile proton was confirmed by the HSQC experiment that showed that no 13C NMR signals correlated with the 1H NMR signal at δH 5.33. The DEPT spectrum classified the 22 carbons into 3 methyl groups, 7 methylene groups, 7 methine groups, and 5 quaternary carbons. Detailed analysis of 1D and 2D NMR spectroscopic data of 5 and a comparison of them with those of tuberastemoninol, a known Stemona alkaloid of tuberostemonine type isolated from Stemona tuberose,[17] revealed not only their very similar spectroscopic features, mainly their identical categories of functional groups, but also noticeable differences in terms of chemical shifts. After the 1HNMR signals were correlated to their corresponding carbon signals by the HSQC experiment, the HMBC experiment confirmed that the constitution of compound 5 was exactly identical with that of the latter compound, which showed the following long-range 1H–13C correlations in 5: from Me-17 to


Magn. Reson. Chem. 2014, 52, 719–728

Structure elucidations of new Stemona alkaloids

Figure 2. Key ROESY correlations for compound 1.




Magn. Reson. Chem. 2014, 52, 719–728

were given in Table 5. Considering the previous information, it was affirmed that the stereoconfiguration of compound 5 was different from that of tuberastemoninol. The relative configuration of 5 was established by the ROESY experiments and starting from the common α-orientation of H-3 in Stemona alkaloids. The NOE correlations of H-3/OH-1, OH-1/H-13, and Me-15/H-11



725

C-10 and C-16; from H-11 to C-1, 9, 14, and C-16; from Me-15 to C12, 13, and C-14; from Me-22 to C-19, 20, and C-21; from H-12 to C-11, 13, 14, and C-15; from H-13 to C-1, 11, 12, 14, and C-15; from H-10 to C-9, 9a, 11, 16, and C-17; from H-3 to C-2, 5, and C-18; from H-2α to C-1, 3, and C-9; from H-2β to C-1, 3, and C-18; and from OH-1 to C-1, 2, and C-9. Other detailed HMBC correlations

Y. Yue et al.


726

indicated the α-orientation of OH-1 and H-13 and the β-orientation of H-11 and H-12. The NOE correlations of H-11/Me-17, H12/Me-17, and H-11/H-16a revealed the α-orientation of H-10. The observation that no NOE correlation was observed between OH-1 and CH2-8 ascertained the relative configuration of C-9 as shown in Fig. 6, i.e. the β-orientation of CH2-8 at C-9. Similarly, the NOE correlation of H-18/H-20 confirmed the cis relationship between H-18 and H-20 in 5. Other detailed NOE correlations were given in Table 5. Thus, the structure of compound 5 was established as (rel)-(3S*,3aS*,3bS*,5S*,10aR*,11R*,11aS*)-11-ethyl3b-hydroxy-3-methyl-5-((2S*,4S*)-4-methyl-5-oxotetrahydrofuran2-yl)decahydro-6,10a-methanofuro[2′,3′:4,5]cyclopenta[1,2-d]azonine2,12(3bH)-dione and was given the trivial name stemonatuberosine A. It has been brought forward that some compounds of Stemona alkaloids can be regarded as artificial. However, to the best of our knowledge, all exemplified artifacts of Stemona alkaloids are dehydroderivatives or bisdehydroderivatives of substrates. With six to seven oxygen atoms existing in the molecules, compounds 1–5 in this paper obviously represent oxidation products of the tuberostemonine series that might derive from this or those substrates of Stemona alkaloids. From the point of view of organic reaction, the conditions of extraction, isolation, and purification in this paper are relatively mild to compounds 1–5 given that there is no intensively powerful oxidant existing that could result in the oxidization to this great extent. Moreover, in view of some facts, such as no carbon–carbon double bond is existent for compounds 1–5 and many other highly oxidized natural compounds have been isolated from the yield of plants up to now, it is uncertain in the case of this paper whether the oxidizing action to compounds 1–5 came from the extraction and fractionation process or the effect of the endogenous mechanism. The isolated compounds were evaluated preliminarily for their AChE-inhibiting and BuChE-inhibiting properties in vitro, with the well-known donepezil and iso-OMPA being used as positive controls, respectively. Acetylthiocholine iodide (ASCh) can be hydrolyzed by AChE to give thiocholine and acetic acid. Thiocholine can react with a thiol reagent, 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), to afford a yellow compound, 1,3,5-trinitrobenzene. According to the colorimetric determination of this resultant, the amount of hydrolyzed thiocholine can be calculated, which is related to the activity of AChE under certain conditions. Likewise, butyrylthiocholine chloride (BuSCh) can be hydrolyzed by BuChE to give thiocholine and butyric acid. With the same procedure as the evaluation of the activity of AChE, the activity of BuChE can also be obtained. In the case of this paper, the inhibition activities of compounds 1, 2, and 5 are shown in Table S1. As one can see, these compounds shared very weak inhibition activities in both experiments.


Materials and methods General The melting point was determined on an XT4-100x microscopic melting point apparatus (Electro-Optical Scientific Instrument Factory, Beijing, China) and was uncorrected. Optical rotations were measured on a PerkinElmer 241 digital polarimeter (PerkinElmer, Waltham, MA, USA) at 20 °C. IR spectra were obtained on a Nicolet 5700 spectrometer (Thermo Electron, Madison, WI, USA). NMR spectra were recorded on a Bruker AVANCE-III 500 NMR spectrometer (500.06 MHz for 1H NMR; 125.75 MHz for 13C NMR, respectively; Bruker Instruments Inc., Germany) with tetramethylsilane (TMS) as internal standard and dimethyl sulfoxide (DMSO)-d6 as test solvent. Preparative HPLC was performed on a Shimadzu LC-6AD system equipped with an SPD-10A detector, and a reversed-phase C18 column (YMC-Pack ODS-A U 20 × 250 mm, 10 μm) was employed. HRESIMS was measured on a Thermo Scientific Exactive Plus Orbitrap (Thermo Fisher Scientific, USA). Column chromatography (CC) was undertaken with silica gel (200–300 mesh, Qingdao Marine Chemical Group Co., Qingdao, China) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). Thin-layer chromatography (TLC) was carried out with glass plate precoated silica gel G. Spots were visualized under UV light and by spraying with Dragendorff’s reagent. Plant material The root of S. tuberosa was collected from Enshi City, Hubei Province, China, in September 2010, and was identified by Associate Professor L. Ma (Institute of Material Medica, Chinese Academy of Medical Sciences and Peking Union Medical College). A voucher specimen (ID-S-2433) has been deposited in the herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Extraction and isolation The dried root of S. tuberosa (19.6 kg) was ground into powder and extracted with 95% ethanol for three times (about five-fold solvent each time) under reflux condition. Combined ethanol extract was evaporated in vacuum to yield a brown, dark residue (2.2 kg), which was suspended in 80–90% aqueous ethanol. The resulting suspension was extracted with petroleum ether for three times in a separatory funnel. Evaporation of the ethanol layer in vacuum yielded another residue which was redissolved in water (8 l), then extracted with EtOAc for three times in a separatory funnel (3 × 4 l). The combined EtOAc phase was washed with the solution of 5% NaHCO3 in H2O for three times


Magn. Reson. Chem. 2014, 52, 719–728

Structure elucidations of new Stemona alkaloids (3 × 4 l) and then with pure H2O for two times (2 × 4 l), respectively, to pH 7. Complete removal of EtOAc in vacuum and over boiling water led to the EtOAc soluble fraction (110 g). The EtOAc soluble fraction was subjected to CC over silica gel and eluted with a gradient mixture of chloroform–methanol (100:0 converting step by step to 1:1), which yielded eight subfractions (designated as F1 to F8) according to their TLC profiles. After the processing of filtration and evaporation of F2 (eluate obtained from CHCl3/MeOH = 100:1), the residue (10 g) was subjected to CC over silica gel using a gradient mixture of petroleum ether/EtOAc (15:1 to 1:1) as eluant, which yielded six subfractions (designated as F2-1 to F2-6). Compound 5 was obtained directly from F2-3 (eluate obtained from petroleum ether/EtOAc = 5:1) as a white amorphous powder (214 mg) by filtration. F3 (15 g, eluate obtained from CHCl3/MeOH = 50:1) was subjected to CC over silica gel using a gradient mixture of petroleum ether/EtOAc (10:1 to 1:1) as eluant, which yielded four subfractions (F3-1 to F3-4). F3-2 (3 g, eluate obtained from petroleum ether/EtOAc = 3:1) was submitted to an ODS-A column eluted with MeOH/H2O of decreasing polarity (40–60% of MeOH in H2O), which yielded two subfractions (F3-2-1 and F3-2-2). Compounds 1 (34 mg) and 2 (76 mg) were isolated from F3-2-1 (750 mg) by preparative reversed-phase high-performance liquid chromatography (RP-HPLC; mobile phase: MeOH–H2O = 60:40; flow rate: 6 ml min1; UV detection at 210 and 230 nm simultaneously), respectively. F3-3 (1 g, eluate obtained from petroleum ether/EtOAc = 2:1) was submitted to an ODS-A column eluted with MeOH/H2O of decreasing polarity (40–60%), which yielded two subfractions (F3-3-1 and F3-3-2). Compounds 3 (6 mg) and 4 (4 mg) were obtained from F3-3-2 (250 mg) by preparative RPHPLC (mobile phase: CH3CN–H2O = 40:60; flow rate: 6 ml min1; UV detection at 210 and 230 nm simultaneously), respectively. NMR data

Magn. Reson. Chem. 2014, 52, 719–728

Characteristics of each alkaloid Stemonatuberone A (=(rel)-(3S*,3aS*,6S*,12R*,13R*,13aS*)-13-ethyl3-methyl-6-((2S*,4S*)-4-methyl-5-oxotetrahydrofuran-2-yl)decahydro-2H-7,12-methanofuro[3,2-e][1]azacyclododecine-2,4,14(8H)trione; 1): white amorphous powder; ½α20 D 22.0 (c 0.10, MeOH); IR (KBr) νmax 2969, 1767, 1704, 1487, 1382, 1230, 1164, 993, and 814 cm1; 1H and 13C NMR data are listed in Table 1; HRESIMS (positive mode) m/z 406.2221[M + H]+ (calcd for C22H32NO6, 406.2224). Stemonatuberone B (=(rel)-(3R*,3aR*,6S*,12R*,13R*,13aR*)-13ethyl-3-methyl-6-((2S*,4S*)-4-methyl-5-oxotetrahydrofuran-2-yl) decahydro-2H-7,12-methanofuro[3,2-e][1]azacyclododecine-2,4,14 (8H)-trione; 2): colorless prismatic crystal; mp 224–226 °C; ½α20 D +13.8 (c 0.10, MeOH); IR (KBr) νmax 2973, 1782, 1709, 1461, 1370, 1258, 1186, 933, and 821 cm1; 1H and 13C NMR data are listed in Table 2; HRESIMS (positive mode) m/z 406.2223[M + H]+ (calcd for C22H32NO6, 406.2224). Stemonatuberone C (=(rel)-(3S*,3aR*,6S*,12R*,13R*,13aS*)-13ethyl-3-methyl-6-((2S*,4S*)-4-methyl-5-oxotetrahydrofuran-2-yl) decahydro-2H-7,12-methanofuro[3,2-e][1]azacyclododecine-2,4,14 (8H)-trione; 3): white amorphous powder; ½α20 D 29.7 (c 0.10, MeOH); IR (KBr) νmax 2957, 1773, 1700, 1627, 1454, 1364, 1233, 1172, 991, and 818 cm1; 1H and 13C NMR data are listed in Table 3; HRESIMS (positive mode) m/z 406.2223[M + H]+ (calcd for C22H32NO6, 406.2224). Stemonatuberonol A (=(rel)-(3S*,3aR*,6S*,12S*,13R*,13aR*)-13ethyl-13-hydroxy-3-methyl-6-((2S*,4S*)-4-methyl-5-oxotetrahydrofuran-2-yl)decahydro-2H-7,12-methanofuro[3,2-e][1]azacyclododecine-2,4,14(8H)-trione; 4): white amorphous powder; ½α20 D +37.7 (c 0.10, MeOH); IR (KBr) νmax 3505, 2980, 1772, 1709, 1648, 1449, 1343, 1254, 1145, 981, and 816 cm1; 1H and 13C NMR data are listed in Table 4; HRESIMS (positive mode) m/z 422.2170[M + H]+, (calcd for C22H32NO7, 422.2173). Stemonatuberosine A (=(rel)-(3S*,3aS*,3bS*,5S*,10aR*,11R*,11aS*)11-ethyl-3b-hydroxy-3-methyl-5-((2S*,4S*)-4-methyl-5-oxotetrahydrofuran-2-yl)decahydro-6,10a-methanofuro[2′,3′:4,5]cyclopenta[1,2d]azonine-2,12(3bH)-dione; 5): white amorphous powder; ½α20 D 4.2 (c 0.10, MeOH); IR (KBr) νmax 3435, 2987, 1758, 1696, 1462, 1378, 1198, 1014, 928, and 768 cm1; 1H and 13C NMR data are listed in Table 5; HRESIMS (positive mode) m/z 406.2227[M + H]+ (calcd for C22H32NO6, 406.2224). AChE-inhibition and BuChE-inhibition assay The assay was performed by an improved DTNB method according to literature.[18] In the enzyme reaction system, the tested compounds (or positive controls) and AChE or BuChE, all with certain concentration (Table S1), were suspended in the reaction buffer. Then, 20 μl of ASCh or BuSCh and DTNB in 0.05 mol l1 phosphate buffer was added. This mixture was incubated for 60 min at 37 °C. The light absorption value, which represented



727

The NMR spectra were recorded at room temperature (298.15 K) on a Bruker AVANCE-III 500 NMR spectrometer (500.06 MHz for 1 H NMR; 125.75 MHz for 13C NMR, respectively) equipped with a 5-mm PA BBOTM probe (Bruker Instruments Inc., Germany). All compounds (about 45 mg each) were dissolved, respectively, in 0.5 ml of DMSO-d6, which contains 0.05% TMS, as the test solvent and transferred into a 5-mm NMR tube. Chemical shifts were referenced to the residual solvent peak of DMSOd6 at 2.49 and 39.5 ppm for proton and carbon, respectively. Coupling constants (J) were in hertz. Data processing was carried out with TOPSPIN 2.1 version (Bruker, Germany). The pulse conditions were as follows: for the 1H NMR spectra, spectrometer frequency (SF) 500.063 MHz, acquisition time (AQ) 3.172 s, relaxation delay (RD) 1.000 s, pulse 90°, spectral width (SW) 10 329.30 Hz, and Fourier Transform (FT) size 64-K data point; for the 13C NMR spectra, SF 125.753 MHz, AQ 1.101 s, RD 1.000 s, pulse 56°, SW 29 583.75 Hz, and FT size 32-K data point; and for the COSY spectra, AQ 0.2642 s, RD 0.889 s, and SW 3164.557 Hz. A sinebell weighting was applied to each dimension and zero filled to 2-K data points. For the HSQC spectra, the conditions are as follows: AQ 0.1321 s, RD 1.000 s, and SW 3164.557 (1H) and 20 704.875 Hz (13C). A sinebell function was applied to the F2 dimension before zero filling to 2-K data points, and a sinebell was applied to the F1 and zero filled to 2-K data points before Fourier transformation. A one-bond coupling constant of 145.0 Hz and long-range coupling constant of 8.0 Hz were used to set delays in the pulse sequence. For the

HMBC spectra, the conditions are as follows: AQ 0.5284 s, RD 0.779 s, and SW 3164.557 (1H) and 25 000.000 Hz (13C). A onebond coupling constant of 145.0 Hz and long-range coupling constant of 8.0 Hz were used to set delays in the pulse sequence. A sinebell weighting was applied to both 1H and 13C dimensions and zero filled to 4 and 1-K data points, respectively; for the ROESY spectra, the conditions are as follows: AQ 0.264 s, RD 0.941 s, SW 3164.557 Hz, and spinlock = 200 ms.

Y. Yue et al. the consumption of ASCh or BuSCh in the reaction system, was recorded at the wavelength of 412 nm. The inhibition rates (IR, %) were obtained by the following formula: IR½%

Acontrol Asample 100 Acontrol Ablank

Acknowledgements This work was supported by grants from the National Science and Technology Project of China (2011ZX09307-002-01 and 2012ZX09301002-002).

References [1] The State Pharmacopoeia Commission of China, The State Pharmacopoeia of the People’s Republic of China, Chinese Medical Science and Technology Press, Beijing, 2010, pp. 123–124. [2] P. Wang, A. L. Liu, Z. An, Z. H. Li, G. H. Du, H. L. Qin. Chem. Biodiv. 2007, 4, 523–530. [3] R. A. Pilli, G. B. Rosso, M. C. F. Oliveira. Nat. Prod. Rep. 2010, 27, 1908–1937. [4] J. Schinnerl, B. Brem, P. P. H. But, S. Vajrodaya, O. Hofer, H. Greger. Phytochemistry 2007, 68, 1417–1427. [5] T. Sastraruji, A. Jatisatienr, S. G. Pyne, A. T. Ung, W. Lie, M. C. Williams. J. Nat. Prod. 2005, 68, 1763–1767.

[6] P. Mungkornasawakul, S. Chaiyong, T. Sastraruji, A. Jatisatienr, C. Jatisatienr, S. G. Pyne, A. T. Ung, J. Korth, W. Lie. J. Nat. Prod. 2009, 72, 848–851. [7] H. Greger, J. Schinnerl, S. Vajrodaya, L. Brecher, O. Hofer. J. Nat. Prod. 2009, 72, 1708–1711. [8] Y. Hitotsuyanagi, H. Fukaya, E. Takeda, S. Matsuda, Y. Saishu, S. Zhu, K. Komatsu, K. Takeya. Tetrahedron 2013, 69, 6297–6304. [9] Y. Gao, J. Wang, C. F. Zhang, X. H. Xu, M. Zhang, L. Y. Kong. Tetrahedron 2014, 70, 967–9744. [10] Y. Zhong, Y. Gao, Q. P. Guo, W. M. Li. Helvet. Chim. Acta 2010, 93, 133–138. [11] H. Greger. Planta Med. 2006, 72, 99–113. [12] R. A. Pilli, M. C. F. Oliveira. Nat. Prod. Rep. 2000, 17, 117–127. [13] P. Wang, A. L. Liu, Z. H. Li, G. H. Du, H. L. Qin. J. Asian Nat. Prod. Res. 2008, 10, 311–314. [14] P. Wang, H. L. Qin, Z. H. Li, A. L. Liu, G. H. Du. Chin. Chem. Lett. 2007, 18, 152–154. [15] Y. Yue, A. J. Deng, D. S. Xu, H. L. Qin. J. Asian Nat. Prod. Res. 2013, 15, 145–150. [16] X. H. Cai, X. D. Luo. Planta Med. 2007, 73, 170–173. [17] W. H. Lin, L. Ma, M. S. Cai, R. A. Barnes. Phytochemistry 1994, 36, 1333–1335. [18] M. Gao, A. L. Liu, G. H. Du. Chin. J. New Drugs 2009, 18, 1145–1148.

Supporting information Additional supporting information may be found in the online version of this article at the publisher’s website.

728 wileyonlinelibrary.com/journal/mrc


Magn. Reson. Chem. 2014, 52, 719–728

A superoxide dismutase purified from the roots from Stemona tuberosa.

Inhibitory effects of Stemona tuberosa on lung inflammation in a subacute cigarette smoke-induced mouse model.

Anti-inflammatory activity of methyl ferulate isolated from Stemona tuberosa Lour.

Stemona tuberosa prevented inflammation by suppressing the recruitment and the activation of macrophages in vivo and in vitro.

Three new diterpenoid alkaloids from the roots of Aconitum duclouxii.

Semen-Like Floral Scents and Pollination Biology of a Sapromyophilous Plant Stemona japonica (Stemonaceae).

Bisbenzylisoquinoline alkaloids from the roots of cyclea tonkinensis.

Two new β-carboline alkaloids from the roots of Gypsophila oldhamiana.

Antitrypanosomal quinoline alkaloids from the roots of Waltheria indica.

Antiviral glycosidic bisindole alkaloids from the roots of Isatis indigotica.

Catharanthus alkaloids XXXII: isolation of alkaloids from Catharanthus trichophyllus roots and structure elucidation of cathaphylline.

New Alkaloids from Aconitum stapfianum.

Diterpene alkaloids from the roots of Aconitum moldavicum and assessment of Nav 1.2 sodium channel activity of aconitum alkaloids.

New potently bioactive alkaloids from Crinum erubescens.

Indole alkaloids from the roots of Isatis indigotica and their inhibitory effects on nitric oxide production.

Alkaloids from the roots of Stichoneuron caudatum and their acetylcholinesterase inhibitory activities.

Four new Amaryllidaceae alkaloids from Zephyranthes candida.

Six new alkaloids from Melodinus henryi.

Two new steroidal alkaloids from Fritillaria ussuriensis.

New Lycopodium alkaloids from Phlegmariurus squarrosus.

New monoterpenoid alkaloids from the aerial parts of Uncaria hirsuta.

Two new alkaloids from the bulbs of Lycoris sprengeri.

New alkaloids from the leaves of Evodia rutaecarpa.

Biosynthesis of bisbenzylisoquinoline alkaloids in cultured roots of Stephania cepharantha.