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Communication

Revised NMR data for Incartine: an Alkaloid from Galanthus elwesii

1
Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat de Barcelona, Av. Diagonal 643, 08028 Barcelona, Spain
2
Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán 04510, México DF, México
*
Author to whom correspondence should be addressed.
Molecules 2007, 12(7), 1430-1435; https://doi.org/10.3390/12071430
Submission received: 18 June 2007 / Revised: 10 July 2007 / Accepted: 10 July 2007 / Published: 12 July 2007

Abstract

:
Phytochemical studies on Galanthus elwesii resulted in the isolation of five alkaloids: incartine, hordenine, hippeastrine, 8-O-demethylhomolycorine and lycorine. The NMR data given previously for incartine were revised and completed by two-dimensional 1H-1H and 1H-13C chemical shift correlation experiments. In vitro studies on the bioactivity of incartine were carried out.

Introduction

Galanthus elwesii (snow drop) is a small bulbous plant distributed throughout South-Eastern European countries and Euro-Asia [1], which has been cultivated for its elegant flowers. Earlier investigations in Galanthus elwesii have resulted in the isolation of a large variety of Amaryllidaceae alkaloids [2,3,4]. Many of these compounds have been found to exhibit among others, strong acetylcholinesterase inhibitory, cytotoxic and antiviral activities [5].
As part of our ongoing studies on the Galanthus species, we report on the isolation, structural elucidation of incartine (1) hordenine (2), lycorine (3), 8-O-demethylhomolycorine (4) and hippeastrine (5) from G. elwesii (Figure 1). Bioactivity of incartine with respect to antiretroviral, cytotoxic and acetylcholinesterase inhibitory activities, hitherto unknown, has also been studied.

Results and Discussion

The EtOH extract from G. elwesii plants was fractioned as described in the Experimental Section and as a result, five alkaloids were obtained.
Figure 1. Chemical structures of the alkaloids isolated from Galanthus elwesii.
Figure 1. Chemical structures of the alkaloids isolated from Galanthus elwesii.
Molecules 12 01430 g001
The HRMS of compound 1, with a parent ion at m/z 333.1572 (calc. mass - 333.1576), suggested the molecular formula C18H23NO5. The complete assignment of the 1H-NMR spectrum (Table 1), performed by 2D COSY and NOESY experiments, revealed a compound with a lycorine type structure whose chemical shifts were closely comparable with those of galanthine [6]. The absence of an olefinic proton in 1, characteristic of the lycorine type compounds [5], as well as the similarity of the chemical shifts of the H-11 and H-12 protons with those of the homolycorine type C3 - C4 epoxy derivatives, galwesine and 9-O-demethylgalwesine [4] indicated a C3 - C4 epoxy ring. On the basis of the 1H-NMR spectral data, 1 was identified as incartine, an alkaloid that has been isolated only once from the flowers of Lycoris incarnata [7]. Compound 1 and incartine have identical MS spectra. However, the 1H-NMR spectrum of 1 was not congruent with that reported in the literature. The H-4a, H-6α, H-6β, H-11β, H-12α and H-12β protons of 1 are considerably shifted, with values of - 0.96, - 0.66, - 0.64, - 0.99, - 1.17, and - 0.88 ppm, respectively, as compared to those described for incartine by Kihara et al. [7]. Moreover, the chemical shifts of compound 1 closely compare with those of the biosynthetically related galwesine [4]. The observed deshielding on the protons in α position surrounding the N atom of the incartine reported by Kihara et al. [7] as compared with 1 as well as the similarities of the 1H-NMR of 1 with those of galwesine and galanthine leads us to suggest that the compound described in the literature is most probably a salt of 1.
Table 1. 1H-NMR, COSY, NOESY and 13C-NMR data for 1.
Table 1. 1H-NMR, COSY, NOESY and 13C-NMR data for 1.
PositionΗ δ (J in Hz)COSYNOESYHMQCHMBC
14.46 br sH-2 H-2, H-10, H-10b, 2-OCH367.7 dC-2, C-3, C4a
23.85 overlappedH-1H-1, H-3, H-4a, 2-OCH378.2 dC-1, C-4, C-10b, 2-OCH3
33.48 brd d (1.0)-H-2, H-11β, 2-OCH359.0 dC-1, C-2
4 66.9 s
4a2.56 d (11.2)H-10bH-2, H-10b61.3 dC-3, C-4, C-6, C-10a
6α3.65 dd (14.0, 1.0)H-6βH-6β, H-7, H-12α56.7 tC-4a, C-6a, C-7, C-12
6β4.07 d (14.0)H-6α H-6α, H-7, H-12β56.7 tC-4a, C-6a, C-7
6a 128.5 s
76.57 s H-6α, H-6β, 8-OCH3110.7 dC-6, C-8, C-9,C-10a, 8-OMe
8 147.8 s
9 147.9 s
106.78 s H-1, 9-OCH3107.6 dC-6a, C-8, C-9, C-10b
10a 127.9 s
10b2.82 d (11.2)H-4aH-1, H-4a, 2-OCH3 40.1 dC-4a, C-10a
11α2.00 ddd (14.5, 6.5, 2.0)H-11β, H-12α, H-12βH-11β, H-12α30.1 tC-3, C-4, C-12
11β2.39 ddd (14.5, 10.3, 7.5)H-11α, H-12α, H-12βH-3, H-11α, H-12β30.1 tC-4, C-4a, C-12
12α2.59 ddd (10.3, 9.0, 6.5)H-11α, H-11β, H-12βH-6α, H-11α, H-12β53.0 tC-4, C-6
12β3.20ddd (9.0, 7.5, 2.0)H-11α, H-11β, H-12αH-6β, H-11β, H-12α53.0 tC-4, C-4a, C-11
2-OMe3.54 s H-1, H-2, H-3, H-10b59.2 qC-2
8-OMe3.80 s H-756.1 qC-8
9-OMe3.84 s H-1056.2 qC-9
The 13C-NMR data of 1, reported here for the first time (Table 1), are in agreement with the proposed structure. The heteronuclear chemical shift correlation experiments (HMBC, HMQC) were performed in order to assign all the signals of the 13C-NMR spectrum and to confirm the assignments made for the 1H-NMR spectrum. Thus, the skeleton of compound 1 contains 18 carbon atoms with the following characteristic signals: (1) three signals at δ 59.2, 56.1, 56.2 for the methoxyl groups at C-2, C-8 and C-9, respectively, (2) three triplets at δ 56.7, 30.1 and 53.0, corresponding to the methylene carbons C-6, C-11 and C-12, (3) two doublets at δ 110.7 and 107.6, assigned to the aromatic carbons C-7 and C-10, and five doublets at δ 67.7, 78.2, 59.0, 61.3, and 40.1, corresponding to the carbons C-1, C-2, C-3, C-4a and C-10b, and (4) five singlets at δ 66.9, 128.5, 147.8, 147.9 and 127.9, assigned to the quaternary carbons C-4, C-6a, C-8, C-9 and C-10a. The 13C-NMR spectrum of compound 1 is in agreement with that reported for galanthine, with the exception of carbons C-3 and C-4 due to the deshielding effect of the double bond ∆3,4 in galanthine [6]. Later examination of its 1H-NMR revealed that compound 1 is unstable and degrades after several months. Compounds 2-5 were identified by comparison of their spectroscopic data with those reported in the literature, as described in the Experimental Section.
A wide range of bioactivities have been reported for compounds 2-5 [5,8]. Alkaloids with the lycorine type skeleton possess significant antiviral and cytotoxic activities. Compound 3 is the most studied alkaloid of this group as it possesses one of the strongest inhibitory properties such as antiviral and cytotoxic among others [6,9,10]. However, incartine showed no remarkable activity against human immunodeficiency virus (HIV) in a human cell culture system or was cytotoxic at the concentrations tested (CC50 42.9 μM/mL). Incartine (IC50 102 μM/mL) and hordenine (IC50 473 μM/mL) inhibited electric eel acetylcholinesterase in vitro considerably less than the positive control galanthamine (5.14 μM/mL).

Experimental

General

1H-, 13C-NMR, COSY, NOESY, HMQC and HMBC spectra were recorded in an Inova 500 MHz using CDCl3 as a solvent and TMS as an internal standard. Chemical shifts were reported in δ units (ppm) and coupling constants (J) in Hz. Mass spectra were recorded on a CG-MS Hewlett Packard 6890+ MSD 5975, (Hewlett Packard, Palo Alto, CA, USA) operating in EI mode at 70 eV. A HP-5 MS column (30 m ± 0.25 mm ± 0.25 μm) was used. The temperature program was: 100-180°C at 15°C·min-1, 1 min hold at 180°C, 180-300°C at 5°C·min-1 and 1 min hold at 300°C. Injector temperature was 280°C. The flow rate of carrier gas (Helium) was 0.8 mL·min-1. Split ratio was 1:20.

Plant material

Whole plants of Galanthus elwesii Hook fil. (Amaryllidaceae) were collected in the flowering stage, in March 2003, from a wild population near the village of Obrochishte, district of Varna, Bulgaria. The plant material was identified by Dr. Ljuba Evstatieva - Institute of Botany, Bulgarian Academy of Sciences (BAS). A voucher specimen was deposited at the Institute of Botany (BAS), with No. SOM-162923.

Extraction and isolation

Fresh G. elwesii plants (2.9 kg) were extracted with 95% ethanol (5 x 10 L, 72 h each). The solvent was evaporated under vacuum and the residue was dissolved in 2% sulfuric acid (300 mL) and defatted with Et2O (5 x 300 mL). Then the acidic soln. was made alkaline with 25% ammonia to pH 9-10, and the alkaloids were extracted with CHCl3 (5 x 500 mL). 2.55 g of crude alkaloid mixture (0.088% referred to fresh weight) were obtained after evaporation of the organic solvent. The crude alkaloid mixture was subjected to column chromatography (5 x 50 cm column) on silica gel (250 g, 40-60 μm, SDS France) using EtOAc gradually enriched with MeOH (100:0, 400 mL; 90:10, 300 mL; 75:25, 300 mL; 50:50, 1000 mL; 20:80, 500 mL and 0:100, 1200 mL). Fractions of 50 mL were collected and monitored by TLC (silica gel, EtOAc/MeOH/25% ammonia 12:3:0.1; Dragendorff’s reagent). Incartine (1, 32 mg) was isolated as a brown solid by CC on silica gel of fraction 24 (40-60 μm; 1 x 30 cm; EtOAc/MeOH 8:2; 5 mL fractions) from sub-fractions 10-12. Hordenine (2, 65 mg) was obtained after crystallization in EtOAc from fractions 39-77. Lycorine (3, 44 mg) crystallized directly from the crude alkaloid mixture. 8-O-Demethylhomolycorine (4, 134 mg) and hippeastrine (5, 158 mg) were purified by pre-crystallization in MeOH from fractions 31-34 and 25-27, respectively. Hordenine (2) was identified by comparison of its spectroscopic data with those already reported [11]. Lycorine (3) [12], 8-O-demethylhomolycorine (4) [13] and hippeastrine (5) [14] were identified by direct comparison of their chromatographic and spectroscopic properties (TLC, CG-MS, MS, 1H-NMR) with those of authentic samples obtained in our laboratory from other plant sources.

Bioactivity assays

Acetylcholinesterase (AChE) inhibitory activity of incartine and hordenine were measured exactly as described by López et al. [15]. Galanthamine was used as a positive control. Antiretroviral activity and cytotoxicity were evaluated in human MT-4 cells based on viability of cells that have been infected or not infected with HIV-1 following exposure to various concentrations of the test compound according to the procedure previously described by López el al. [16]. AZT and nevirapine were used as positive controls.

Acknowledgments

This work was partially financed by the Generalitat de Catalunya (2005SGR-00020). Both R. Reyes-Chilpa and S. Berkov thank the Spanish Ministerio de Educación y Ciencia for a research fellowship. The authors are grateful to Dr. S. López for technical assistance with acetylcholinesterase assays and Dr. J.A. Esté for the antiretroviral and cytotoxic assays.

References

  1. Webb, D.A. Flora Europaea; Tutin, T.G., Heywood, V.H., Burges, N.A., Moore, D., Valentine, D.H., Walters, S.M., Webb, D.A., Eds.; Cambridge University Press: Cambridge, U.K., 1980; Vol. 5; p. 77. [Google Scholar]
  2. Boit, H.; Horst, E. Amaryllidaceae alkaloids VIII. Alkaloids from Sprekelia formosissima, Galanthus elwesii, Zephyranthes candida, and Crinum powellii. Chem. Ber. 1955, 88, 1590–1594. [Google Scholar] [CrossRef]
  3. Cherkasov, O. Plant sources of galanthamine. Khim.-Farm. Zh. 1977, 11, 84–87. [Google Scholar]
  4. Latvala, A.; Önür, M.; Gözler, T.; Linden, A.; Kivçak, B.; Hesse, M. Alkaloids of Galanthus elwesii. Phytochemistry 1995, 39, 1229–1240. [Google Scholar] [CrossRef]
  5. Bastida, J.; Lavilla, R.; Viladomat, F. The Alkaloids; Cordell, G., Ed.; Elsevier Inc.: Amsterdam, The Netherlands, 2006; Vol. 63; pp. 87–179. [Google Scholar]
  6. Bastida, J.; Codina, C.; Viladomat, F.; Rubiralta, M.; Quirion, J.-C.; Husson, H.-P.; Ma, G. Narcissus alkaloids XIII. Complete assignment of the NMR spectra of papyramine and 6-epi-papyramine by two-dimensional NMR spectroscopy. J. Nat. Prod. 1990, 53, 1456–1462. [Google Scholar]
  7. Kihara, M.; Xu, L.; Konishi, K.; Kida, K.; Nagao, Y.; Kobayashi, S.; Shingu, T. Isolation and structure elucidation of a novel alkaloid, incartine, a supposed biosynthetic intermediate, from flowers of Lycoris incarnata. Chem. Pharm. Bull. 1994, 42, 289–292. [Google Scholar] [CrossRef]
  8. DNP: Dictionary of Natural Products; The Chapman &Hall/CRC, Web Version 2004. Available online: http://www.chemnetbase.com/scripts/dnpweb.exe Accessed September 06, 2006.
  9. Likhitwitayawuid, K.; Angerhofer, C.; Heebyung, C.; Pezzuto, J.; Cordell, G. Cytotoxic and antimalarial alkaloids from the bulbs of Crinum amable. J. Nat. Prod. 1993, 56, 1331–1338. [Google Scholar] [CrossRef]
  10. Szlávik, L.; Gyuris, A.; Minárovits, J.; Forgo, P.; Molnár, J.; Hohmann, J. Alkaloids from Leucojum vernum and antiretroviral activity of Amaryllidaceae alkaloids. Planta Med. 2004, 70, 871–873. [Google Scholar] [CrossRef]
  11. Tato, M.P.V.; Castedo, L.; Riguera, R. New alkaloids from Pancratium maritimum. Heterocycles 1988, 27, 2833–2838. [Google Scholar]
  12. Bastida, J.; Fernández, J.; Viladomat, F.; Codina, C.; Fuente, G. Alkaloids from Narcissus tortuosus. Phytochemistry 1995, 38, 549–551. [Google Scholar]
  13. Bastida, J.; Llabrés, J.; Viladomat, F.; Codina, C.; Rubiralta, M.; Feliz, M. 9-O-demethyl-maritidine: A new alkaloid from Narcissus radinganorum. Planta Med. 1988, 54, 524–526. [Google Scholar] [CrossRef]
  14. Almanza, G.; Fernández, J.; Wakori, E.; Viladomat, F.; Codina, C.; Bastida, J. Alkaloids from Narcissus cv. Salome. Phytochemistry 1996, 43, 1375–1378. [Google Scholar] [CrossRef]
  15. López, S.; Bastida, J.; Viladomat, F.; Codina, C. Acetylcholinesterase activity of some Amaryllidaceae alkaloids and Narcissus extract. Life Sci. 2002, 71, 2521–2529. [Google Scholar] [CrossRef]
  16. López, S.; Armand-Úgon, M.; Bastida, J.; Viladomat, F.; Esté, J.A; Stewart, D.; Codina, C. Anti-human immunodeficiency virys type 1 (HIV-1) activity of lectines from Narcissus species. Planta Med. 2003, 69, 109–112. [Google Scholar] [CrossRef]
  • Sample Availability: Samples of compounds 2 - 5 and reference NMR spectra of 1-5 are available from the authors.

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MDPI and ACS Style

Berkov, S.; Reyes-Chilpa, R.; Codina, C.; Viladomat, F.; Bastida, J. Revised NMR data for Incartine: an Alkaloid from Galanthus elwesii. Molecules 2007, 12, 1430-1435. https://doi.org/10.3390/12071430

AMA Style

Berkov S, Reyes-Chilpa R, Codina C, Viladomat F, Bastida J. Revised NMR data for Incartine: an Alkaloid from Galanthus elwesii. Molecules. 2007; 12(7):1430-1435. https://doi.org/10.3390/12071430

Chicago/Turabian Style

Berkov, Strahil, Ricardo Reyes-Chilpa, Carles Codina, Francesc Viladomat, and Jaume Bastida. 2007. "Revised NMR data for Incartine: an Alkaloid from Galanthus elwesii" Molecules 12, no. 7: 1430-1435. https://doi.org/10.3390/12071430

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