Alkaloids from Crinum erubescens Aiton

1 Introduction

Plants belonging to the Amaryllidaceae family are well known for containing an exclusive group of alkaloids, which have been considered a distinctive taxonomic characteristic of this family (Bastida et al., 2006). Crinum is a pantropical genus that has extensive traditional use in Africa in a wide range of therapeutic applications, including antitumoral and antimalarial, and treatment of rheumatism and kidney and bladder infections, among others (Nair and van Staden, 2013). Alkaloids purified from the Crinum species have been confirmed as responsible for some of these properties, particularly the antiproliferative action (Berkov et al., 2011a). The alkaloid-rich extracts from Crinum angustum have shown significant antibacterial and antifungal activities (Ianello et al., 2014a). To date, approximately 130 species found throughout Africa, America, southern Asia and Australia have been classified within the Crinum genus (Refaat et al., 2012).

GC–MS has proven to be a useful tool in the identification and quantification of Amaryllidaceae alkaloids. This technique has been successfully used to assist with the isolation of new structures from alkaloid-rich extracts by comparing their component electron impact-mass fragmentation spectra (EI-MS) with those of known standards. Such a guided approach has been extensively applied as part of a global Amaryllidaceae Phytochemical Program, in which our research group is closely involved, and whose main focus is to characterize the alkaloid profiles of these species. Within this framework, the anti-Trichomonas vaginalis agent candimine and the acetylcholinesterase (AChE) inhibitory 11β-hydroxygalanthamine have been recently isolated from Brazilian Amaryllidaceae species (de Andrade et al., 2011; Giordani et al., 2010), and the anti-Alzheimer’s disease (AD) drug, galanthamine, has been quantified in several natural sources (Berkov et al., 2008a, 2011b; Torras-Claveria et al., 2013). The global program has also led to the characterization of several new compounds from Leucojum aestivum (Berkov et al., 2008b), Pancratium illyricum (Iannello et al., 2014b), and others. All together, the obtained results represent a valuable contribution to the understanding of chemical and biological aspects of Amaryllidaceae alkaloids.

In the present work, the phytochemical study of the indigenous Costa Rican Crinum erubescens led to the identification of eight alkaloids using an in-home GC–MS database. Some of them have been previously isolated from the same species (Wildman et al., 1967; Fales et al., 1959; Wildman and Bailey, 1968). In particular, the new compound 1-epidemethylbowdensine (1) was completely characterized by NMR, while its absolute configuration was established by CD spectroscopy.

2 Experimental

3 Results and discussion

The known alkaloids bowdensine (2), 1-epidemethoxybowdensine (3), crinamine (4), macronine (5), 11,12-dehydroanhydrolycorine (6), ismine (7) and trisphaeridine (8) were identified by means of GC–MS (Table 1). Macronine (5) was the major component present in C. erubescens, and a significant relative percentage of crinamine (4) was also found. The GC–MS technique cannot provide the absolute stereochemistry of the Amaryllidaceae alkaloids, although the previous isolation of various bowdensine variants and crinamine in C. erubescens was taken into account for the identification of compounds 2, 3 and 4 (Fales et al., 1959; Wildman et al., 1967; Wildman and Bailey, 1968). The structures of the alkaloids identified in C. erubescens in the present work are shown in Fig. 1. The GC–MS chromatogram of the alkaloid-rich fractions is shown in Fig. 2.

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Figure 1

Alkaloids identified from Crinum erubescens.

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Figure 2

GC–MS chromatogram of n-Hex (A), EtOAc (B) and EtOAc:MeOH (C) extracts from Crinum erubescens with the identified alkaloids.

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The GC–MS data for 1 were similar to those observed for the 5,10b-ethanophenanthridine derivatives (Bastida et al., 2006; Duffield et al., 1965). Its HRESIMS suggested the molecular formula C₂₀H₂₄NO₇ for the parent ion [M + H]⁺ at m/z 390.1553 (calcd 390.1547). The EI-MS spectra showed the molecular ion peak [M⁺] at m/z 389 (100%) and two major fragments at m/z 330 [M-59]⁺ and m/z 270 [M-119]⁺, which are typical for the loss of two vicinal acetoxy groups (Viladomat et al., 1996). Characteristic ¹H NMR signals included the following: (1) one aryl singlet at δ 6.13, confirmed as H-10 due to the NOESY correlation with H-1; (2) two doublets (J = 1.5) at δ 5.87–5.86, typical of the methylenedioxy group; (3) a d at δ 5.34 (J = 4.4) and a dt at δ 5.58 (J = 5.5, 4.0) assigned to H-1 and H-2, respectively, which were shifted to a lower field due to the presence of 1- and 2-acetoxy substituents. The magnitude of the coupling constant in H-1 and H-2 resonances confirmed that both 1- and 2-acetoxy substituents were β-oriented, as in 1-epibowdensine (Viladomat et al., 1996); (4) two AB doublets at δ 4.29 and 3.86, corresponding to the benzylic protons at C-6, with H-6α being assigned to a lower field due to its cis relationship with the nitrogen lone pair. All remaining NMR data, including NOESY and HMBC experiments (Fig. 3), were in agreement with the structure of a new bowdensine derivative 1-epidemethylbowdensine (1).

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Figure 3

Key NOESY ( $\leftrightarrow$ ) and HMBC ( $\to$ ) correlation of compound 1.

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Although the basic crinane structure of 1 was well established by 1D and 2D NMR experiments, the absolute stereochemistry of the 5,10b-ethanophenanthridine alkaloids should be confirmed by CD spectroscopy. Indeed, the shape, amplitude and sign of the CD spectra are crucial for the correct orientation of the 5,10b ethano bridge in crinane variants (De Angelis and Wildman, 1969; Wagner et al., 1996). The CD spectrum of 1 displayed a positive and negative Cotton effect at ca. 250 and 290 nm, respectively, which is characteristic of a crinine-type compound (Wagner et al., 1996). Therefore, by means of NMR and CD spectroscopy, the compound 1-epidemethylbowdensine (1) was completely characterized and its NMR data are shown in Table 2.

1-Epidemethylbowdensine (1): amorphous solid; [α]_D²⁴ + 18.7 (c 0.23, CHCl₃); CD [Θ]²⁰_λ: [Θ]₂₄₈ + 703.95, [Θ]₃₀₀ – 58.90; UV (MeOH) λ_max(log ε) 240 (3.39), 276 (2.99) nm; IR (CHCl₃) ν_max 2924, 2854, 1743, 1625, 1480, 1376, 1247, 1036, 946, 757 cm⁻¹; EIMS data are shown in Table 1; ¹H NMR (CDCl₃, 500 MHz) and ¹³C NMR (CDCl₃, 125 MHz) are detailed in Table 2; HRESIMS of [M + H]⁺ m/z 390.1553 (calcd for C₂₀H₂₄NO₇, 390.1547).

4 Conclusions

The phytochemical investigation of C. erubescens, native to Costa Rica, led to the identification of eight alkaloids by GC–MS. A complete spectroscopic characterization, including absolute configuration determined by CD, of the new compound 1-epidemethylbowdensine has been performed, leading to its confirmation as a new crinine-type derivative.