Alkaloid Constituents of the Amaryllidaceae Plant Amaryllis belladonna L.

The plant family Amaryllidaceae is well-known for its unique alkaloid constituents, which exhibit a wide range of biological activities. Its representative, Amaryllis belladonna, has a geographical distribution covering mainly southern Africa, where it has significant usage in the traditional medicine of the native people. In this study, A. belladonna samples collected in Brazil were examined for alkaloid content. Alkaloid profiles of A. belladonna bulbs were generated by a combination of chromatographic, spectroscopic and spectrometric methods, including GC–MS and 2D NMR. In vitro screening against four different parasitic protozoa (Trypanosoma cruzi, T. brucei rhodesiense, Leishmania donovani and Plasmodium falciparum) was carried out using the A. belladonna crude methanol extract, as well as three of its alkaloid isolates. Twenty-six different Amaryllidaceae alkaloids were identified in the A. belladonna bulb samples, and three of them were isolated. Evidence for their respective biosynthetic pathways was afforded via their mass-spectral fragmentation data. Improved data for 1-O-acetylcaranine was provided by 2D NMR experiments, together with new 1H-NMR data for buphanamine. The crude extract and 3-O-acetylhamayne exhibited good antiprotozoal activity in vitro, although both with a high cytotoxic index.


Introduction
The plant family Amaryllidaceae has attracted considerable attention in view of the fascinating structural features and varied biological activities manifested by its alkaloid principles. Anticancer, antimicrobial and anticholinesterase activities represent some of these biological properties [1]. The genus Amaryllis L. comprises two species, A. belladonna and A. acuminata, both native to southern Africa [2]. Also referred to by the folk names "belladonna-lily" and "naked-lady", A. belladonna has been used for several centuries in the medicinal traditions of the Sotho, Xhosa and Zulu peoples of South Africa, and in Java for the treatment of "swelling" (a presumed synonym for cancer) [2,3].
The characteristic isoquinoline alkaloids produced by members of the Amaryllidaceae are derived from the aromatic amino acids phenylalanine and tyrosine, which combine to give the common precursor O-methylnorbelladine [4]. Consequently, alternative ways of oxidative phenolic
Based on their structural identities, it has been possible to show how the alkaloids identified in A. belladonna could be linked biosynthetically ( Figure 1). We propose that the chemical similarity of the alkaloid types from this species may be due to different combinations of common biosynthetic reactions, such as hydroxylation, hydrogenation, reduction, epoxidation, methylation, methoxylation, acetylation and allylic rearrangements, starting from a common precursor. Precedence for these reactions has been studied in other alkaloid groups; for example, indole alkaloids in Catharanthus roseus (Apocynaceae) [26,27].
To date, galanthamine metabolization represents the most studied Amaryllidaceae alkaloid biosynthetic pathway. Experiments involving the application of 13 C-labelled O-methylnorbelladine to field-grown Leucojum aestivum plants indicate that galanthamine biosynthesis involves phenolic oxidative coupling of O-methylnorbelladine to a dienone, the adduct of which undergoes spontaneous conversion to N-demethylnarwedine. The latter is converted via stereoselective reduction to N-demethylgalanthamine, which is then N-methylated to galanthamine [10].
Benzylamine O-methylnorbelladine (9) was also shown to be a precursor of norpluviine (27), which is known to be integral to lycorine-type alkaloid metabolization [28]. Apart from this, the transformation of caranine (5) into lycorine (15) has been observed in both Zephyranthes candida and Clivia miniata [29,30]. In A. belladonna, it is conceivable that 1-O-acetylcaranine (6) and 1-O-acetyllycorine (17) are derived via acetylation of the C-1 hydroxy group in caranine (5) and lycorine (15), respectively. Furthermore, we suggest that lycorine (15) could be metabolized to anhydrolycorine (4) by double dehydration of the C-1 and C-2. Also, we suggest that anhydrolycorine (4) may be a precursor of 11,12-dehydroanhydrolycorine (10) by reduction of the D-ring double bond, which could then serve as a precursor to hippadine (19) via hydroxylation at C-6 and subsequent oxidation to the amide functionality.
Based on their structural identities, it has been possible to show how the alkaloids identified in A. belladonna could be linked biosynthetically ( Figure 1). We propose that the chemical similarity of the alkaloid types from this species may be due to different combinations of common biosynthetic reactions, such as hydroxylation, hydrogenation, reduction, epoxidation, methylation, methoxylation, acetylation and allylic rearrangements, starting from a common precursor. Precedence for these reactions has been studied in other alkaloid groups; for example, indole alkaloids in Catharanthus roseus (Apocynaceae) [26,27]. To date, galanthamine metabolization represents the most studied Amaryllidaceae alkaloid biosynthetic pathway. Experiments involving the application of 13 C-labelled O-methylnorbelladine to field-grown Leucojum aestivum plants indicate that galanthamine biosynthesis involves phenolic

1-O-Acetylcaranine
The base ion signal that occurs at m/z 252 in 1-O-acetylcaranine (6) is characteristic of deacetylation at C-1 and dehydration at C-1 and C-2 ( Figure 2). Herein we present the complete NMR data for 1-O-acetylcaranine (6) by both 1D and 2D NMR spectroscopic analysis (see 1-O-acetylcaranine NMR spectra in Supplementary Materials). The 1 H-NMR spectrum ( Table 2) was similar to that of 1-O-acetylcaranine and caranine [13,31,32]. The shift of the H-1 proton signal to a lower magnetic field than that observed for caranine suggested a substitution of the hydroxyl group at C-1, which was further substantiated by the presence of a singlet at δ 1.93, indicative of an acetyl group. The 13 C-NMR spectrum showed a singlet resonance signal at δ 171.0, which confirmed the presence of one carbonyl group. The COSY spectrum showed an allylic coupling between H-3 and H-4a and between H-3 and H-11, which allowed us to determine the H-10b proton location in the 1 H-NMR spectrum. In addition, the small magnitude of the coupling constants between H-1 and H-10b allowed us to assign the α-orientation to the acetyl group. The two C-6 protons were differentiated as an AB system with a geminal coupling of around 14 Hz. H-4a showed NOESY correlations with both H-2α and H-6α, which turned out to be key correlations in the assignment of their orientation. Furthermore, H-12α was ascribable to a higher field as a consequence of NOESY contour correlation with H-6α. The HMBC spectrum allowed us to assign the quaternary carbons C-6a (δ 129.5) and C-10a (δ 127.8) via three-bond correlation with H-10 and H-7, respectively. The aromatic protons were ascribable to H-7 and H-10 due to three-bond HMBC correlations with C-10a and C-6a, respectively, in addition to NOESY correlations observed for the H-6/H-7 and H-1/H-10 proton pairs. The C-8 quaternary carbon (δ 146.2) was located via four-bond HMBC connectivity with both H-6, as well as via three-bond connectivity with H-10. The quaternary carbon C-9 (δ 146.5) was determined via three-bond HMBC connectivity with H-7. Finally, the singlet resonance signal at δ 139.26 was assigned to C-4, taking into account three bond connectivities to H-12β. All these data are in agreement with the structure of 1-O-acetylcaranine (6) (see Figure 4). bond HMBC connectivity with H-7. Finally, the singlet resonance signal at δ 139.26 was assigned to C-4, taking into account three bond connectivities to H-12β. All these data are in agreement with the structure of 1-O-acetylcaranine (6) (see Figure 4).

Buphanamine
For most crinine-type alkaloids, the molecular ion is prominent as the base peak, and the fragmentation mechanism is initiated by opening of the C-11/C-12 ethano-bridge, indicating bond cleavage at the position β to the nitrogen atom ( Figure 3) [33].

Buphanamine
For most crinine-type alkaloids, the molecular ion is prominent as the base peak, and the fragmentation mechanism is initiated by opening of the C-11/C-12 ethano-bridge, indicating bond cleavage at the position β to the nitrogen atom ( Figure 3) [33]. The 1 H-NMR data we obtained for buphanamine (16) (Table 3) differed slightly from the data available in the literature [34]. The discrepancies we noted relate to the H-4α, H-4β, H-4a, H-6α, H-6β, H-11endo, H-11exo, H-12endo and H-12exo proton resonances (Δ +0.26, +0.22, +0.36, +0.26, +0.27, +0.14, +0.20, +0.24, +0.51, respectively). These are quite downfield-shifted compared to the corresponding data we obtained in our NMR analysis (see buphanamine NMR spectra in Supplementary Materials). Quaternization of the nitrogen atom via N-oxide or salt formation is known to influence the chemical shifts of protons in its vicinity [1].   The 1 H-NMR data we obtained for buphanamine (16) (Table 3) differed slightly from the data available in the literature [34]. The discrepancies we noted relate to the H-4α, H-4β, H-4a, H-6α, H-6β, H-11endo, H-11exo, H-12endo and H-12exo proton resonances (∆ +0.26, +0.22, +0.36, +0.26, +0.27, +0.14, +0.20, +0.24, +0.51, respectively). These are quite downfield-shifted compared to the corresponding data we obtained in our NMR analysis (see buphanamine NMR spectra in Supplementary Materials). Quaternization of the nitrogen atom via N-oxide or salt formation is known to influence the chemical shifts of protons in its vicinity [1]. In the 1 H-NMR spectrum, the coupling constants between H-1 and H-2 (J = 5.5 Hz), H-2 and H-3 (J = 10.0 Hz), H-3 and H-4α (J = 4.5 Hz), together with the geminal coupling of around 19.7 Hz between H-4α and H-4β, allowed us to place the hydroxyl group at C-1 and the double bond between C-2 and C-3. The NOE contour between H-1 and 2H-11, and the homoallylic coupling between H-1 and H-4β, confirmed the α-orientation for the hydroxyl group. Interestingly, the COSY correlation observed between H-2 and 2H-4 also confirmed the allylic coupling between these protons. The two H-6 protons were clearly differentiated as part of an AB system, each with a geminal coupling value of 17.2 Hz. The singlet aromatic proton resonance was ascribable to H-10 due to three-bond HMBC correlations with C-6a and C-10b, in addition to a NOESY correlation with H-1, allowing the aromatic methoxyl group to be placed at C-7. H-6α was assigned to a lower field via a NOESY contour correlation with H-4a (Figure 4). The H-11exo and H-12exo protons were assignable based on the large values of their respective coupling constants. The quaternary carbons C-6a and C-10a were ascribed by means of their three-bond HMBC correlations with H-10 and H-1, respectively. Finally, the singlet resonance signal at δ = 48.3 was assigned to C-10b, taking into account three-bond connectivities to H-10, H-4α and H-4β. The absolute configuration of this alkaloid was determined from the CD spectrum, wherein the curve was qualitatively similar to that of buphanamine (16) [35]. and H-4β, confirmed the α-orientation for the hydroxyl group. Interestingly, the COSY correlation observed between H-2 and 2H-4 also confirmed the allylic coupling between these protons. The two H-6 protons were clearly differentiated as part of an AB system, each with a geminal coupling value of 17.2 Hz. The singlet aromatic proton resonance was ascribable to H-10 due to three-bond HMBC correlations with C-6a and C-10b, in addition to a NOESY correlation with H-1, allowing the aromatic methoxyl group to be placed at C-7. H-6α was assigned to a lower field via a NOESY contour correlation with H-4a ( Figure 4). The H-11exo and H-12exo protons were assignable based on the large values of their respective coupling constants. The quaternary carbons C-6a and C-10a were ascribed by means of their three-bond HMBC correlations with H-10 and H-1, respectively. Finally, the singlet resonance signal at δ = 48.3 was assigned to C-10b, taking into account three-bond connectivities to H-10, H-4α and H-4β. The absolute configuration of this alkaloid was determined from the CD spectrum, wherein the curve was qualitatively similar to that of buphanamine (16) [35].

Biological Activity
The biological activity tests against the parasitic protozoa and for cytotoxicity were performed as described earlier [36]. 3-O-Acetylhamayne showed higher activity than the other alkaloids against all protozoan parasites tested. It was active against Trypanosoma brucei rhodesiense (IC 50 = 1.51 µg mL −1 ), T. cruzi (IC 50 = 8.25 µg mL −1 ), Leishmania donovani (IC 50 = 17.91 µg mL −1 ) and Plasmodium falciparum (IC 50 = 1.14 µg mL −1 ). However, the cytotoxicity against L6 cells (rat skeletal myoblasts, IC 50 = 1.72 µg mL −1 ) highlights that 3-O-acetylhamayne is not a selective antiparasitic agent. The crude extract exhibited activity against T. brucei rhodesiense (IC 50 = 4.67 µg mL −1 ), T. cruzi (IC 50 = 34.86 µg mL −1 ) and P. falciparum (IC 50 = 1.17 µg mL −1 ), but it was less cytotoxic than 3-O-acetylhamayne. There is very little information about the structure-antiprotozoal activity relationship of the Amaryllidaceae alkaloids, but some results suggest that the methylenedioxy group can contribute to increase the antiprotozoal activity in these alkaloids [24]. The crude extract results suggest that A. belladonna could be an interesting source of alkaloids with antiparasitic activity. The antiprotozoal activity of A. belladonna alkaloids is summarized in Table 4. The extracts were subjected to a combination of chromatographic techniques, including vacuum liquid chromatography (VLC) [25] and semi-preparative thin-layer chromatography (TLC). The general VLC procedure consisted of the use of a silica gel 60 A (6-35 µm) column with a height of 4 cm and a variable diameter according to the amount of sample (2.5 cm for 400-1000 mg; 1.5 cm for 150-400 mg). Alkaloids were eluted with n-hexane containing increasing EtOAc concentrations, followed by neat EtOAc, which was gradually enriched with MeOH (reaching a maximum concentration of 20% v/v). Fractions of 10-15 mL were collected, monitored by TLC (UV 254 nm, Dragendorff's reagent) and combined according to their profiles. For semi-preparative TLC, silica gel 60F 254 was used (20 cm × 20 cm × 0.25 mm) together with different solvent mixtures depending on each particular sample (EtOAc:MeOH, 9:1 v/v, or EtOAc:MeOH, 8:2 v/v), always in an environment saturated with ammonia. The alkaloids were each identified by GC-MS and the three known alkaloids were isolated and structurally elucidated by NMR as 1-O-acetylcaranine (362.9 mg), 3-O-acetylhamayne (2.2 mg) and buphanamine (17.2 mg).   Table 3; ESI-MS data shown in Table 1