Revised Nmr Data for Incartine: an Alkaloid from Galanthus Elwesii

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 1 H-1 H and 1 H-13 C 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].

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.The HRMS of compound 1, with a parent ion at m/z 333.1572 (calc.mass -333.1576),suggested the molecular formula C 18 H 23 NO 5 .The complete assignment of the 1 H-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 1 H-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 1 H-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 1 H-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.The 13 C-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 13 C-NMR spectrum and to confirm the assignments made for the 1 H-NMR spectrum.Thus, the skeleton of compound 1 contains 18 carbon atoms with the following characteristic signals:  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 13 C-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 1 H-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 (CC 50 42.9μM/mL).Incartine (IC 50 102 μM/mL) and hordenine (IC 50 473 μM/mL) inhibited electric eel acetylcholinesterase in vitro considerably less than the positive control galanthamine (5.14 μM/mL).

Experimental
General 1 H-, 13 C-NMR, COSY, NOESY, HMQC and HMBC spectra were recorded in an Inova 500 MHz using CDCl 3 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.

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.