New Ceramides from Acnistus arborescens

The genus Acnistus (Solanaceae) comprises 50 tropical American species of shrubs and small trees distributed from Mexico to Southern Argentina. Plants of this genus biosynthesize a complex group of natural C 28 steroidal lactones, known as acnistins, withanolides, and jaborols. As part of a collaborative search program to identify novel naturally occurring anticancer agents, we have investigated Acnistus arborescens (L.) Schlecht. The plant presents different therapeutic uses in the traditional medicine, for instance, the hot infusion of leaves and barks is used in the treatment of bruises and sprains, moreover, the leaves have been used to treat liver and spleen diseases, and cancerous growths. Indeed, previous studies on this species have led to the isolation of several cytotoxic withanolides. Recently, it was published for the first time the isolation of withaphysalins, including its cytotoxic effects against several tumor cell lines. In this paper, the isolation and characterization of two new ceramides from A. arborescens is described. This is the first examples of ceramides from a plant of the Acnistus genus and, to the best of our knowledge, the second report about such kind of secondary metabolites from the Solanaceae family. Ceramides and related compounds have been isolated extensively from fungi and several marine organisms such as sponges, tunicates, sea stars, green algae and gorgonians. However, more recently, this class of compound has been isolated from some higher plants.


Introduction
The genus Acnistus (Solanaceae) comprises 50 tropical American species of shrubs and small trees distributed from Mexico to Southern Argentina. 1 Plants of this genus biosynthesize a complex group of natural C 28 steroidal lactones, known as acnistins, 2 withanolides, 3 and jaborols. 4s part of a collaborative search program to identify novel naturally occurring anticancer agents, we have investigated Acnistus arborescens (L.) Schlecht. 5,6The plant presents different therapeutic uses in the traditional medicine, for instance, the hot infusion of leaves and barks is used in the treatment of bruises and sprains, moreover, the leaves have been used to treat liver and spleen diseases, and cancerous growths. 1,7,8Indeed, previous studies on this species have led to the isolation of several cytotoxic withanolides. 7,8ecently, it was published for the first time the isolation of withaphysalins, including its cytotoxic effects against several tumor cell lines. 5,6n this paper, the isolation and characterization of two new ceramides from A. arborescens is described.This is the first examples of ceramides from a plant of the Acnistus genus and, to the best of our knowledge, the second report about such kind of secondary metabolites from the Solanaceae family. 9eramides and related compounds have been isolated extensively from fungi 10 and several marine organisms such as sponges, 11 tunicates, 12 sea stars, 13 green algae 14 and gorgonians. 15[18]

Results and Discussion
The EtOH extracts from leaves and stems of A. arborescens were fractionated by column chromatography on silica gel by elution with n-hexane, CH 2 Cl 2 , EtOAc and MeOH.After several chromatographic procedures compound 1 was isolated from the leaves, while compound 2 was obtained from the stems.The two compounds, after detailed spectroscopic analysis, were identified as two new ceramides.
Compound 1 was isolated as a white amorphous solid.Its IR spectrum revealed the absorption bands for either secondary amides or hydroxyl groups at 3302 cm −1 , an amide carbonyl at 1638 cm −1 and skeletal absorptions at 1547, 1518 and 1464 cm −1 for aromatic rings.The molecular formula of  -8) characteristic of the methylene attached to the amide nitrogen and a triplet at d 2.97 (J 7.5 Hz, 2H-7) for the benzyl methylene.As expected, the COSY spectrum showed vicinal scalar coupling for the former methylene with the benzyl methylene and the amide hydrogen.Additionally, the 1 H NMR spectrum showed a signal at d 2.41 (t, J 7.5 Hz, 2H-2') typical of methylene group a to a carbonyl, as well as signals for a terminal methyl group at 0.89 (t, J 6.5 Hz, 3H-28') and a series of aliphatic methylene hydrogens (d 1.39-1.26).The 13 C NMR spectra of 1 showed the presence of a carbonyl signal at d 174.Accordingly with the afore mentioned spectral data, the structure of 1 was characterized as the N-(4-hydroxyphenethyl) octacosamide, an unknown ceramide up to date.
Compound 2 was also isolated as a white amorphous solid.Its IR spectrum revealed several broad peaks in the range of 3399-3227 cm −1 characteristic of bonded N-H or O-H stretching, the amide carbonyl at 1621 cm −1 , followed by the N-H bending at 1544 cm −1 .A shoulder at higher frequency than the carbonyl absorption followed by the band at 964 cm −1 is in accordance with a trans carbon-carbon double bond.The long aliphatic chain was characterized by a band at 723 cm −1 .The molecular formula of 2, C 40 H 79 NO 5 , was determined on the basis of highresolution ESI mass spectroscopy in the negative mode ([M-H] − ) at m/z 652.5983 (C 40 H 78 NO 5 , calc.652.5880).The 1 H NMR spectrum showed characteristic signal for an amide proton at d 8.60 (d, J 8.9 Hz), resonances for four hydroxyl groups at d 7.63, 6.72 (integrating for two hydrogens) and 6.10, all appearing as broad singlet; a signal at d 5.15 (m, H-2) for a methine bonded to a nitrogen; signals at d 4.54 (dd, J 10.9 and 4.5 Hz, H-1a) and 4.44 (dd, J 10.9 and 4.8 Hz, H-1b) for a hydroxymethylene, as well as signals at d 4.64 (H-2´), 4.38 (m, H-3) and 4.32 (m, H-4) corresponding to three oxymethines.Additionally, signals for a double bond at d 5.59 (td, J 15.4 and 5.4 Hz, H-16) and 5.50 (td, J 15.4 and 5.8 Hz, H-17), two terminal methyl at d 0.88 (t, J 6.2 Hz, 3H-19´/3H-21) and several methylene hydrogens at d 2.29-1.28corresponding to two aliphatic chains were also observed.The COSY spectrum revealed coupling for the methine attached to the nitrogen (H-2) with the oxymethylene (2H-1) and an oxymethine (H-3) protons, and the latter with the oxymethine H-4.As expected, the 13   (C-1), 77.2 (C-3) and 73.5 (C-4).Furthermore, HMBC correlation between the proton signal at 4.64 (H-2´) and the carbonyl (C-1') confirmed the presence of a side chain of a a-hydroxy fatty acid.Subsequently, the four hydroxyl groups were confirmed through the acetylated compound (2a  S10 (Supplementary Information), were congruent with the proposed structure.After comparison with analogous compounds [16][17][18] the relative stereochemistry inferred for the stereocenters 2, 3, 4 and 2' was presumed to be S*, S*, R* and R*, respectively.On the basis of the above mentioned data, the structure of compound 2 was established as rel-(2S,3S,4R,16E)-2-[(2'R)-2'-hydroxynonadecanoylamino]heneicosadec-16-ene-1,3,4-triol.

General experimental procedures
Melting points were measured on a digital Mettler Toledo FP90 apparatus and are uncorrected.The optical rotations were measured on a Perkin-Elmer 341 digital polarimeter.IR spectra were recorded using a Perkin-Elmer FT-IR 1000 spectrometer.Electrospray ionization-High resolution mass spectra were measured on a quadrupole-time of flight instrument (UltrOTOF-Q, Bruker Daltonics, Billerica, MA), while the low resolution Electron ionization mass spectra were acquired on a Shimadzu QP5050A instrument, through direct probe and operating at 70 eV.All NMR experiments were performed on a Bruker Avance DRX-500 spectrometer equipped with a 5 mm inverse detection z-gradient probe. 1 H NMR (500.13MHz) and 13
C NMR spectra of 2 exhibited three downfield carbon signals at d 175.7 (C-1'), 131.3 (C-16) and 131.2 (C-17) corresponding to a carbonyl amide and a double bond, respectively.Signals for a nitrogenated methine at d 54.4 (C-2), an oxymethylene at d 62.5 (C-1)and three oxymethynes at d 77.2 (C-3), 73.5 (C-4) and 72.9 (C-2').In addition, several carbon signals in the range of d 36.2-23.4related to methylene groups and a carbon signal at 14.8 corresponding to two terminal methyls were also deduced from13 C NMR spectra, suggesting that compound 2 was also a ceramide.The unequivocal positions of the hydroxyl groups were deduced based on the HMBC spectrum in which the proton signal at d 8.60 (NH) showed correlations with the carbonyl (C-1') and the nitrogenated methine (C-2), while the proton signal at d 5.15 (H-2) exhibited correlations with the carbon signals at d 62.5

Figure 1 .
Figure 1.Structures of compounds 1, 2 and the acetylated derivative 2a.Key long-range correlations observed in the HMBC spectrum of 2.
1, C 36 H 65 NO 2 , was determined on the basis of high-resolution ESI mass spectrometry in the negative mode ([M-H] − ) at m/z 542.4907 (C 36 H 64 NO 2 , calc.542.4937).The 1 H NMR spectrum of 1 ).The HMQC spectrum showed the obvious additional methyl signals at d H /d C 2.23/21.2,2.13/21.1,2.10/21.5 and 2.08/21.3characteristic of the four acetyl moieties.Through the HMBC spectra of both compounds 2 and 2a the double bond was assigned at C-16 and C-17 of the sphingoid chain.Hz) displayed between H-16 and H-17.The low resolution mass spectrum of 2 was crucial to the definition of the amide and of the sphingoid portions length.The peaks at m/z 384, 357 and 339 were in agreement with the sphingoid moiety when combined with the peaks at m/z 298 and 280, represented by the structural fragments sketched in Figure C NMR (125.77MHz) spectra were measured at 27 °C using pyridine-d 5 as solvent.Chemical shifts, given on the d scale, were referenced to the residual pyridine [d H 8.74, 7.58, 7.22; d C 150.35, 135.91, 123.87].Column chromatography was run using silica gel 60 (70-230 mesh, Vetec; 230-400 mesh, Merck) and TLC was performed on precoated silica gel polyester sheets (Kieselgel 60 F 254 , 0.20 mm, Merck) by detection with a spray reagent of vanillin/perchloric acid/ EtOH solution followed by heating at 100 °C.

Table 1 .
13 and13C NMR spectral data for compounds 1 and 2, in pyridine-d 5 a,b Signals can be interchangeable.