Alkaloids from Stemona mairei

• Abstract
• Materials and Methods
• Supporting information
• Acknowledgements
• References

Abstract

Three new alkaloids, maireistemoninol (1), neotuberostemonone (2) and epoxytuberostemonone (3), together with six known alkaloids, neotuberostemoninol (4), neotuberostemonine (5), bisdehydroneotuberostemonine (6), bisdehydrotuberostemonine (7), 2-oxostenine (8), and stemotinine (9), were isolated from the roots of Stemona mairei (levl.) Krause. The structures were elucidated by a combination of 1D, 2D-NMR, and mass spectral techniques.

Plants of the genus Stemona (Stemonaceae) have been used in China as insecticides and a cough remedy [1]. About eighty Stemona alkaloids, as unique secondary metabolites in the family Stemonaceae, have been isolated [2]. Some of them reportedly have insecticidal, antitussive, and anti-inflammatory activities [3], [4], [5]. However, up to now, few of the alkaloids were reported in Stemona mairei [6], which is locally distributed in Shichuan and the North of the Yunnan province [7], P. R. China. In our investigation, three new alkaloids, named maireistemoninol (1), neotuberostemonone (2) and epoxytuberostemonone (3), along with six known alkaloids, neotuberostemoninol (4) [8], neotuberostemonine (5) [9]. bisdehydroneotuberostemonine (6) [9], bisdehydrotuberostemonine (7) [9], [10], 2-oxostenine (8) [11], [12], and stemotinine (9) [13], were isolated from the roots of S. mairei (Fig. [1]). In this paper, we describe the isolation and structural elucidation of these alkaloids on the basis of spectroscopic techniques (for the spectral data of the known alkaloids, see the Supporting Information).

Maireistemoninol (1) was obtained as colorless needle crystals. Assignment of the molecular formula as C₂₂H₃₁NO₆ was based on the HR-ESI-MS data (m/z = 428.2045 [M + Na]⁺, calcd.: 428.2049). Its IR (KBr) spectrum showed absorption bands for hydroxy (3477 cm^-1) and carbonyl groups (1762, 1760, and 1669 cm^-1). The ¹H-NMR spectrum indicated the presence of a primary methyl group at δ_H = 1.02 (3H, t, J = 7.5 Hz), two secondary methyl groups at δ_H = 1.15 (3H, d, J = 7.5 Hz) and 1.18 (3H, d, J = 7.5 Hz), and four low-field protons attached to carbon atoms bearing an oxygen or nitrogen at δ_H = 4.70 (1H, t, J = 3.7 Hz), 4.58 (1H, m), 4.53 (1H, m) and 4.06 (1H, brs). The ¹³C-MR and DEPT spectra of 1 showed 22 carbon atoms: three lactonic carbonyl atoms [δ_C = 186.5 (s), 179.1(s) and 178.5(s)], nine methine carbons [δ_C = 80.9 (d), 78.4 (d), 77.0 (d), 74.2 (d), 50.3 (d), 44.7 (d), 43.9 (d), 42.5 (d), and 35.9 (d)], six methylene groups [δ_C = 33.6 (t), 32.0 (t), 31.8 (t), 30.1 (t), 20.2 (t), and 18.4(t)], three methyl groups [δ_C = 15.5 (q), 12.6 (q), and 11.0 (q)], and a quaternary carbon [δ_C = 51.4, (s)]. The ¹³C-NMR and DEPT patterns of 1 were identical to those of the known compound tuberostemoninol, having the same molecular formula. Compared to the ¹³C-NMR spectrum of tuberostemoninol [14], signals for one of the methylenes and for the quaternary carbons [δ_C = 81.7 (s)] disappeared in 1; instead, additional two methines were present in 1 suggesting that the position of the hydroxy group in 1 was different from that in tuberostemoninol.

In the HMBC specrum of 1, the signal at δ_H = 4.06 (1H, brs) corresponding to δ_C = 77.0 (d) in the HSQC spectrum correlated with δ_C = 31.8 (C-7, t), 51.4 (C-9, s), 186.5 (C-9a, s), and 44.7 (C-10, d) (Table [1]) thereby placing the hydroxy group at C-8, which was confirmed by cross-peaks between H-8 (δ_H = 4.06, brs) and H-7 [δ_H = 1.92 (m), 1.68 (m)], H-6 (δ_H = 1.46, m), H-5 [δ_H = 1.75 (m), 1.65 (m)] in the TOCSY spectrum. Its ROESY spectrum suggested that H-8, H-11, H-12, H-13 and H-18 were β-orientated due to correlations [H-11 (δ_H = 4.70, t, J = 3.7 Hz) with H-13 (δ_H = 3.04, m), H-12 (δ_H = 2.47, m), and H-17 (δ_H = 1.02, t, J = 7.5 Hz); H-8 with H-16 (δ_H = 1.68, m); H-12 with H-18 (δ_H = 4.58, m)] and because of the 10-ethyl β-orientation in Stemona-type alkaloids. The α-orientation of H-1 was proposed based on ROESY cross-peaks: H-12/H-2β (δ_H = 1.78, m), H-2α (δ_H = 2.15, m)/H-1(δ_H = 2.85, m). Moreover,  ROESY correlations [H-18/H-12 and H-20 (δ_H = 2.73, m), H-20/H-19β (δ_H = 2.00, m), H-19α (2.50, m)/H-3 (4.53, m)] indicated that the α-methyl-γ-lactone ring was attached to C-3 in a β-orientation together with an α-configuration of Me-20 (Fig. [2] and Table [1]). The full assignments and connectivities were determined by ¹H-¹H COSY, HSQC, HMBC, TOCSY and HMQC-TOCSY spectra.

Neotuberostemonone (2) has a molecular formula of C₂₂H₃₁NO₆ as deduced from HR-ESI-MS at m/z = 428.2047 [M + Na]⁺ (calcd.: 428.2049). The characteristic cleavage fragment m/z = 306 [M - C₅H₇O₂]⁺ in the EI-MS indicated that 3 has an α-methyl-γ-lactone ring connected to C-3 of the A ring [9]. The ¹H- and ¹³C-NMR spectra of 2 were very similar to those of tuberostemonone except for the chemical shifts of rings A, B and C [10], which suggested that the stereo-configurations of the compounds were different. ROESY correlations in 2 among H-11 (δ_H = 4.54, t, J = 3.5 Hz), H-12 (δ_H = 3.56, m), H-13 (δ_H = 2.81, m), H-17 (δ_H = 0.96, t, J = 7.5 Hz), as well as H-9 (δ_H = 2.74, m) indicated that H-9, H-11, H-12 and H-13 are all β-oriented. ROESY correlations from H-19β to H-11 and H-20, from H-19α to H-22, from H-18 to H-2β, H-20, and H-5β, from H-2α (δ_H = 2.04, m) to H-3 (δ_H = 4.85, m) indicated that H-3 and Me-20 were α-orientated (Fig. [2]).

Compound 3 was found to possess a molecular formula of C₂₂H₂₉NO₇ based on the HR-ESI-MS data (m/z = 442.1835 [M + Na]⁺, calcd.: 442.1841). The ¹H-NMR, ¹³C-NMR and DEPT spectra of 3 were similar to those of 2 except for two methines changing to two quaternary carbons [δ_C = 68.0 (s) and 66.1 (s)]. The two quaternary carbons were assigned to a 9,10-epoxide oxygen which was demonstrated by HMBC correlations [C-10 (δ_C = 68.5, s) with H-8 (δ_H = 1.67, m), H-11 (δ_H = 4.26, d, J = 7.6 Hz), H-12 (δ_H = 3.21, t, J = 7.6 Hz), and H-17 (δ_H = 1.04, t, J = 7.5 Hz); C-9 (δ_C = 66.1, s) with H-7 (δ_H = 1.92 and 1.70, m) and H-16 (δ_H = 2.19 and 1.43, m] (Table [1]). The ROESY correlations of 3 [H-15 (δ_H = 1.33, d, J = 7.5 Hz)/H-12 and H-17, H-11/H-13 (δ_H = 2.79, m)] and the fact that no correlation between H-11 and H-12 was observed indicated that H-12 and H-15 were both β-oriented, while H-11, H-13, and the 9,10-epoxide oxygen possessed α-orientations. In addition, the relative configuration of H-3 was deduced as α based on the finding that H-19α (δ_H = 1.66, m) correlated with H-3 (δ_H = 5.21, m) and H-13 in the ROESY spectrum (Fig. [2]).

Materials and Methods

General: All the melting points were obtained on an XRC-1 micromelting apparatus and are uncorrected. IR (KBr) spectra were obtained on a Bio-Rad FTS-135 infrared spectrophotometer (Bio-Rad; Hercules, CA, USA). The NMR spectra (¹H, ¹³C, DEPT, ROESY, ¹H-¹H COSY, HMQC and HMBC) were obtained on Bruker AM-400 and DRX-500 MHz spectrometers (Bruker; Fällanden, Switzerland) with TMS as an internal standard. MS data were obtained on a VG Autospec-3000 spectrometer (VG; Manchester; UK). Column chromatography was performed with silica gel (Qingdao Meigao Chemical Group Co. Ltd; Qingdao, China). TLC was performed on silica gel GF254 plates, and spots were detected by spraying with Dragendorf’s reagent.

Plant material: The roots of S. mairei were collected from Lijiang, Yunnan province, People’s Republic of China. A voucher specimen (No. 0 311 006) has been deposited in the Herbarium of the Department of Taxonomy, Kunming Institute of Botany, the Chinese Academy of Sciences.

Extraction and isolation: Dried roots of S. mairei (9.0 kg) were cut and refluxed with 95 % EtOH (3 × 15 L). After removal of the EtOH under reduced pressure, the viscous concentrate was acidified with dilute (2 %) HCl and then partitioned with CHCl₃ (3 × 3 L). The water layer was adjusted to pH = 9 - 10 with NaOH solution and extracted with CHCl₃ (3 × 3 L) again. Crude 5 was crystallized from the concentrated CHCl₃ solution and further recrystallized from the MeOH solution. The mother liquor was concentrated under vacuum to yield 45 g of residue, of which 40 g were absorbed by silica gel (50 g) and subjected to column chromatography over silica gel (5 × 100 cm, 500 g), eluting with petroleum ether-Me₂CO [9 : 1 (6 L), 8 : 2 (9 L), 7 : 3 (6 L), 6 : 4 (5 L), 1 : 2 (8 L)] to give 4 fractions (I - IV). Fraction II (14 g) was purified by column chromatography over silica gel (4 × 75 cm, 300 g) developed with petroleum ether-Me₂CO [9 : 1 (5 L), 17 : 3 (4 L), 8 : 2 (5 L)] to afford compound 5 (50 mg), 2-oxostenine (6 mg) and a mixture of 6 and 7 (100 mg). Fraction III (7 g) was loaded on CC over silica gel (3 × 60 cm, 100 g) to give subfractions A and B using petroleum ether-Me₂CO [7 : 3 (4 L)]. Subfraction A (3.2 g) was further chromatographed on RP-18 (40 - 63 μm, 3 × 45 cm) eluted with MeOH-H₂O [7 : 3 (1.2 L), 8 : 2 (1.6 L)] to afford compounds 1 (7 mg) and 3 (11 mg), respectively. Compound 8 (23 mg) was obtained from fsubraction B (1.4 g) also by RP-18 with MeOH-H₂O. Fraction IV (6 g) was also subjected to RP-18 silica gel (40 - 63 μm, 3 × 45 cm) to yield compounds 4 (5 mg) and 2 (23 mg) using MeOH-H₂O [6 : 4 (1.5 L), 7 : 3 (1.5 L), 8 : 2 (2.0 L)] as eluent.

Maireistemoninol (1): C₂₂H₃₁NO₆, white needle crystals, m. p. 155 - 157 °C; [α]_D ²⁴: 98.48° (c 0.24, CHCl₃); IR (KBr): ν_max = 3477, 1762, 1760, 1669 cm^-1; ¹H- and ¹³C-NMR data, see Table [1]; EI-MS: m/z = 406[M + H]⁺ (1), 389 (5), 371 (10), 360 (100), 342 (45), 332 (80), 290 (55), 262 (75); HR-ESI-MS: m/z = 428.1599 [M + Na]⁺ (calcd. for C₂₂H₃₁NO₆Na: 428.2049).

Neotuberostemonone (2): C₂₂H₃₁NO₆, white square crystals, m. p. 228 - 230 °C; [α]_D ²⁴: 129.63° (c 0.38, CHCl₃); IR (KBr): ν_max = 1779, 1776, 1691, 1640 cm^-1; ¹H- and ¹³C-NMR data, see Table [1]; EI-MS: m/z = 405 [M]⁺ (4), 306 [M - C₅H₇O₂]⁺ (100); HR-ESI-MS: m/z = 428.2047 [M + Na]⁺ (calcd. for C₂₂H₃₁NO₆Na: 428.2049).

Epoxytuberostemonone (3): C₂₂H₂₉NO₇, white needle crystals, m. p. 286 - 288 °C; [α]_D ²⁴: -67.53° (c 0.16, CHCl₃); IR (KBr): ν_max = 3434, 1780, 1715, 1664 cm^-1; ¹H- and ¹³C-NMR data, see Table [1]; EI-MS: m/z = 419 [M]⁺ (65), 363 (10), 320 [M - C₅H₇O₂]⁺ (25), 292 (30), 252 (50); HR-ESI-MS: m/z = 442.1835 [M + Na]⁺ (calcd. for C₂₂H₂₉NO₇Na: 442.1841).

Supporting information

1D and 2D-NMR spectra of the known compounds are available as Supporting Information.

Acknowledgements

The authors are grateful to the National Natural Science Foundation of China (30 670 214), Natural Science Foundation of Yunnan (grant no. 2004C0009Z) and the Chinese Academy of Sciences (XiBuZhiGuang Project) for financial support and to members of the analytical group in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, for the spectral measurements.

References

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  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Xiao-Dong Luo

State Key Laboratory of Phytochemistry and Plant Resources in West China

Kunming Institute of Botany

Chinese Academy of Sciences

Heilongtan

Kunming 650204

People’s Republic of China

Phone: +86-871-522-3188

Fax: +86-871-515-0227

Email: xdluo@mail.kib.ac.cn

References

• 1 Jiangsu New Medical College. Dictionary of Chinese Traditional Medicine (in Chinese). Shanghai; Shanghai Science and Technology Publisher 1977 Vol. 1: 858-61
  Search in Google Scholar
• 2 Greger H. Structural relationships, distribution and biological activities of Stemona alkaloids.  Planta Med. 2006;  72 99-113
  Thieme ConnectPubMedSearch in Google Scholar
• 3 Pilli R A, Oliveira M CF. Recent progress in the chemistry of the Stemona alkaloids.  Nat Prod Rep. 2000;  17 117-27
  CrossrefPubMedSearch in Google Scholar
• 4 Jiwajinda S, Hirai N, Watanabe K, Santisopasri V, Chuengsamarnyart N, Koshimizu K. et al . Occurrence of the insecticidal 16,17-didehydro-16(E)-stemofoline in Stemona collinsae .  Phytochemistry. 2001;  56 693-5
  CrossrefPubMedSearch in Google Scholar
• 5 Brem B, Seger C, Pacher T, Hofer O, Vajrodaya S, Greger H. Feeding deterrence and contact toxicity of Stemona alkaloids-a source of potent natural insecticides.  J Agric Food Chem. 2002;  50 6383-8
  CrossrefPubMedSearch in Google Scholar
• 6 Lin W H, Ye Y, Xu R S. Studies on new alkaloids of Stemona mairei .  Chin Chem Lett. 1991;  2 369-70
  PubMedSearch in Google Scholar
• 7 Zhan H J, Duyfjes B EE. Flora of China. Beijing; Science Press 1997 Vol. 24: 71
  Search in Google Scholar
• 8 Jiang R W, Hon P M, But P PH, Chung H S, Lin G, Ye W C. et al . Isolation and stereochemistry of two new alkaloids from Stemona tuberosa .  Tetra-hedron. 2002;  58 6705-12
  CrossrefPubMedSearch in Google Scholar
• 9 Ye Y, Qin G W, Xu R S. Alkaloids from Stemona tuberosa .  Phytochemistry. 1994;  37 1201-3
  CrossrefPubMedSearch in Google Scholar
• 10 Lin W H, Ye Y, Xu R S. Chemical studies on new Stemona alkaloids, IV. Studies on new alkaloids from Stemona tuberosa .  J Nat Prod. 1992;  55 571-6
  CrossrefPubMedSearch in Google Scholar
• 11 Gotz M, Bogri T, Gray A H, Strunz G M. The structure of tuberostemonine.  Tetrahedron. 1968;  24 2631-43
  CrossrefPubMedSearch in Google Scholar
• 12 Uyeo S, Irie H, Harada H. The structure of stenine, a new alkaloid occurring in Stemona tuberose .  Chem Pharm Bull. 1967;  15 768-70
  PubMedSearch in Google Scholar
• 13 Xu R S, Lu Y L, Chu J H, Iwashita T, Naaoki H, Naya Y. et al . Studies on some new Stemona alkaloids.  Tetrahedron. 1982;  38 2667-70
  CrossrefPubMedSearch in Google Scholar
• 14 Lin W H, Ma L, Cai M S, Barnes R A. Two minor alkaloids from roots of Stemona tuberosa .  Phytochemistry. 1994;  36 1333-5
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Xiao-Dong Luo

State Key Laboratory of Phytochemistry and Plant Resources in West China

Kunming Institute of Botany

Chinese Academy of Sciences

Heilongtan

Kunming 650204

People’s Republic of China

Phone: +86-871-522-3188

Fax: +86-871-515-0227

Email: xdluo@mail.kib.ac.cn

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