Alkaloids from Hippeastrum papilio

Galanthamine, an acetylcholinesterase inhibitor marketed as a hydrobromide salt (Razadyne®, Reminyl®) for the treatment of Alzheimer’s disease (AD), is obtained from Amaryllidaceae plants, especially those belonging to the genera Leucojum, Narcissus, Lycoris and Ungernia. The growing demand for galanthamine has prompted searches for new sources of this compound, as well as other bioactive alkaloids for the treatment of AD. In this paper we report the isolation of the new alkaloid 11β-hydroxygalanthamine, an epimer of the previously isolated alkaloid habranthine, which was identified using NMR techniques. It has been shown that 11β-hydroxygalanthamine has an important in vitro acetylcholinesterase inhibitory activity. Additionally, Hippeastrum papilio yielded substantial quantities of galanthamine.


Introduction
The object of many studies, Amaryllidaceae alkaloids contain a wide range of chemical structures and interesting biological properties [1], showing pronounced antimalarial [2], antitumoral [3] and acetylcholinesterase inhibitory activity [4]. The use of galanthamine in palliative therapy for mild-OPEN ACCESS moderate AD [5] has prompted the search for analogous compounds bearing the galanthamine-type skeleton. Additionally, as most of the galanthamine used in clinics is supplied from natural sources, there is considerable interest in finding new Amaryllidaceae species for a sustainable production of galanthamine [4,6].
Plants of the genus Hippeastrum, which is endemic to South America, have yielded interesting bioactive compounds such as montanine, with significant psychopharmacological and acetylcholinesterase inhibitory activity [7,8] and candimine, active against Trychomonas vaginalis [9]. Recent nrDNA ITS sequences data have included it within the Hippeastroid subclade and alluded to a probable Brazilian origin [10].
We have identified six known alkaloids, including significant quantities of galanthamine which was the main alkaloid isolated, from the bulbs and leaves of Hippeastrum papilio (Ravenna) Van Scheepen, which grows in Southern Brazil. Furthermore, we have clarified the correct position of the hydroxyl-substituent in the alkaloid habranthine through the isolation of its epimer, the new alkaloid 11β-hydroxygalanthamine. Habranthine was isolated from Pancratium maritimum without certainty about the stereochemistry of the hydroxyl substituent at position 11 in the galanthamine-type skeleton. Using modern 2D NMR, we report the correct assignment of 11β-hydroxygalanthamine, a new alkaloid from Hippeastrum papilio, confirming that the previously reported habranthine is in fact 11αhydroxygalanthamine. Furthermore, 11β-hydroxygalathamine has demonstrated a good ability to inhibit the acetylcholinesterase enzyme, with IC 50 of 14.5 ± 0.33 µM.

Results and Discussion
Bulbs and leaves of the plant showed similar alkaloid profiles by analytical TLC. After Vaccum Liquid Chromatography (VLC) and a purification process, the new alkaloid, 11β-hydroxygalanthamine (1), as well as six known alkaloids, namely galanthamine (2), which was found in relatively high quantities, narwedine (3), haemanthamine (4), 11-hydroxyvittatine (5), 8-O-demethylmaritidine (6) and vittatine (7), were isolated and identified by NMR, CD and MS spectrometry ( Figure 1). Earlier studies have reported the isolation of galanthamine and other galanthamine-type alkaloids from Hippeastrum species, including European cultivars, but mainly as minor compounds.  Compound 1, 11β-hydroxygalanthamine, crystallized as white needles. EI-MS showed a molecular ion peak at m/z 303. The base peak at m/z 230 evidenced the loss of the hydroxyl group at C-3 and the C-11/C-12/NMe residue, in agreement with other galanthamine-type alkaloids [11]. The 1 H-NMR of compound 1 (Table 1) was very similar to that of habranthine previously isolated from Habranthus brachyandrum and Pancratium maritimum [12,13], with small differences in the chemical shifts of H-11 and H-1. The key to the assignation was the large coupling constant J (11α,12β) = 10.8 Hz, observed in compound 1, which indicates a trans diaxial relationship between H-11α and H-12β, and therefore a β-position for the hydroxyl substituent. In contrast, the coupling constants observed for the H-11 of habranthine (J (11β,12β) = 1.6 Hz) and (J (11β,12α) = 4.5 Hz) indicated that the hydroxyl-substituent should be in the α-position (endo). Moreover, the H-12 protons in compound 1 are clearly separated in a double doublet, and 2D analysis confirmed the correct assignment, where NOESY correlations between H-12β and H-4/H-6β and between H-12α and NMe were observed. The complete assignment of the 11β-hydroxygalanthamine is presented in Table 1.
Alkaloid 1 has also proven to be an inhibitor of acetylcholinesterase like the majority of the galanthamine-type alkaloids. In an acetylcholinesterase inhibition screening of several Amaryllidaceae alkaloids, López et al. [14] found that habranthine, epimer of compound 1, showed similar activity to galanthamine. The β-configuration of the hydroxyl group at position 11 in 1 could be unfavourable for its interactions within the active site of the acetylcholinesterase enzyme. The IC 50 for 1 was 14.5 ± 0.33 µM, while galanthamine showed an IC 50 of 1.18 ± 0.07 µM.

General
NMR spectra were recovered in a Varian Mercury 400 MHz instrument using CDCl 3 (CD 3 OD for compound 5) as a solvent and TMS as the internal standard. Chemical shifts were reported in δ units (ppm) and coupling constants (J) in Hz. EIMS were obtained on a GC-MS Hewlett-Packard 6890+ MSD 5975 operating in EI mode at 70 Ev.
An 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 and 180-300 °C at 5 °C min -1 and 10 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. A QSTAR Elite hybrid Quadrupole-Time of Flight (QToF) mass spectrometer (Applied Biosystems, PE Sciex, Concord, ON, Canada) was used for HR-MS analysis. ToF MS data were recorded from m/z 70 to 700 amu with an accumulation time of 1 s and a pause between the mass range of 0.55 ms, operating in the positive mode. Reserpine (1 ρmol/µL) in product ion scan mode of m/z 609 was used for calibration of the mass spectrometer. Optical rotations were carried out on a Perkin-Elmer 241 polarimeter. A Jasco-J-810 Spectrophotometer was used to run CD spectra, all recorded in MeOH. UV spectra were obtained on a DINKO UV2310 instrument and IR spectra were recorded on a Nicolet Avatar 320 FT-IR spectrophotometer.

Microplate AChE Assay
The assay for measuring AChE activity was performed as described by López et al. [14]. Galanthamine hydrobromide was used as a positive control. The IC 50 of 11β-hydroxygalanthamine, galanthamine hydrobromide and galanthamine was measured in triplicate and the results are presented as a mean ± standard deviation using the software package Prism (Graph Pad Inc., San Diego, USA). Both isolated alkaloids and the positive control were evaluated at a concentration ranging from 10 -3 M to 10 -8 M.

Conclusions
Although the chemical synthesis of galanthamine has been achieved, its supply for clinical use still comes from natural sources. Hippeastrum papilio is able to produce great quantities of galanthamine but more studies on its genetic improvement, hybridization or in vitro culture are needed. Our search for new sources of galanthamine and acetylcholinesterase inhibitors has resulted in the isolation and identification of 11β-hydroxygalanthamine (1), a new acetylcholinesterase inhibitor alkaloid. The compound was structurally elucidated by 2D NMR, which allowed us to distinguish it from its epimer, habranthine. Galanthamine-type alkaloids are well-known for their inhibitory activity of the acetylcholinesterase enzyme. The action of galanthamine as an allosterically potentiating ligand in nicotinic acetylcholine receptors [5] and its ability to inhibit β-amyloid aggregation [19] could also play a role in successful AD therapy. The discovery of new galanthamine-type candidates is therefore of real interest for the future management of this disease.