Three New Steroidal Glycoalkaloids from Solanum pseudoquina A . St .-Hil . ( Solanaceae )

Three new steroidal glycoalkaloids (SGA) were isolated from green berries of Solanum pseudoquina A. St.-Hil. The extract obtained after treatment with 5% acetic acid aqueous solution was subjected to several chromatographic procedures, leading to an enriched alkaloidal fraction. The enriched alkaloidal fraction was subjected to a semi preparative high performance liquid chromatography (HPLC) leading to the isolation of three new SGA from S. pseudoquina: 3-O-(β-D-glucopyranosyl) (20S,25S)-22,26-epimino-16α-acetyl-cholesta-22(N)-ene, 3-O-(β-D-glucopyranosyl) (20R,25ξ)-23,26-epimino-16α-acetyl-cholesta-5,23(N)-dien-22-one and 3-O-(β-D-glucopyranosyl) (20S,25ξ)-23,26-epimino-16α-acetyl-cholesta-5,23(N)-dien-22-one. The structures of the new compounds were elucidated with the help of 1D and 2D nuclear magnetic resonance (NMR) and infrared spectroscopy together with high resolution electrospray ionization and atmospheric pressure chemical ionization mass spectrometry.


Introduction
Solanum pseudoquina A. St.-Hil.belongs to the Solanaceae family, and is popularly known as "quina de São Paulo".This plant is endemic in Brazil, and its bark is traditionally used as tonic and febrifuge in the Brazilian southern region. 1 Many species of bitter tasting plants, as S. pseudoquina, have been used in Brazil as substitutes of Cinchona spp., the source of quinine, used in the treatment of intermittent fevers. 2 Plants of the genus Solanum, with approximately 1400 species, are widespread in the tropical and temperate zones.The genus Solanum is the predominating taxon for steroidal alkaloids (SA) within the Solanaceae. 3 Usubillaga et al. 4 reported the isolation, from S. pseudoquina, of the alkaloid solaquidine (3,3-dimethoxy-22β,25ξ-22,26epimino-5ξ-cholestane).In 1987, the same author used partial synthesis to establish the absolute configuration of the natural compound as 5α, 22S and 25R. 5 Later, the isolation from S. pseudoquina of the steroidal alkaloid (25S)-isosolafloridine as well as the demonstration of its convulsive effect were reported, 6 but no further studies with the alkaloidal fractions of S. pseudoquina were reported since then.Steroidal alkaloids and their glycoside derivatives (steroidal glycoalkaloids, SGA) have been isolated from a comprehensive number of Solanum plants.For example, the toxicity of SGA from blighted, green, or sprouted potato (S. tuberosum) tubers (solanine and chaconine), is well documented 7 and has been motivating the research efforts on plant breeding in order to reduce SGA levels in potatoes.Additional interest regarding steroidal alkaloids is due to its antifungal and anticancer activities: solasodine rhamnosyl glycosides as solamargine and solasonine, specifically induce apoptosis in cancer cells and these cells do not develop resistance to this class of compounds. 8n the present report we communicate the isolation of three new steroidal glycoalkaloids from S. pseudoquina: )-23,26-epimino-16α-acetyl-cholesta-5,23(N)dien-22-one, 2 and its isomer 3-O-(β-D-glucopyranosyl) (20R,25ξ)-23,26-epimino-16α-acetyl-cholesta-5,23(N)dien-22-one, 3. The first compound is an isosolafloridine steroidal type alkaloid lacking a double bond between C-5 and C-6. 9 The other two compounds are isomeric solaspiralidine steroidal type alkaloids similar to the steroidal alkaloid isolated from roots of Solanum spirale by Ripperger. 10The three compounds were isolated from the aqueous acidic extract of berries of S. pseudoquina and were structurally characterized by 1D and 2D nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectrometry (HRESIMS), atmospheric pressure chemical ionization mass spectrometry (APCIMS) along with infrared spectroscopy (IR) and optical rotation measurements.

General procedures
KBr pellet IR spectra were recorded on an IRPrestige-21 Shimadzu FTIR spectrophotometer (Kyoto, Japan).Optical rotations were measured in a P-2000 Jasco polarimeter (Tokyo, Japan) using methanol as solvent.NMR spectra were recorded on a Varian VNMRSYS 500 MHz spectrometer (Varian Inc., Palo Alto, CA, USA) working at 499.78 ( 1 H) and at 125.68 MHz ( 13 C).The pulse sequences used are all standard in the VNMRJ software, and the experiments were conducted at 25 °C.Samples were dissolved in 0.3 mL of pyridine-d 5 (1) or CD 3 OD (2 and 3) and transferred to 3 mm NMR tubes.Mass analyses were carried out on a Bruker MicroTOF II (Bruker Daltonics, Bremen, Germany) mass spectrometer equipped with both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) interfaces and controlled by COMPASS software.All samples were dissolved in methanol.For all HRESIMS analyses the parameters were: flow of 180 mL h -1 ; positive ion polarity; capillary set 4500 V; dry gas flow of 5.0 L min -1 and nebulizer at 0.5 Bar.For all APCIMS experiments the parameters were: flow of 0.150 mL min -1 ; positive ion polarity; capillary set 4000 V; dry gas flow of 3.0 L min -1 and nebulizer at 2.5 Bar.Semi preparative high performance liquid chromatography (HPLC) was carried out using two parallel LC-10AS pumps, coupled to a SPD-10A UV-Vis detector, a CTO-10A column oven and a C-R6A integrator (Shimadzu, Kyoto, Japan).

Plant material
Fruits from S. pseudoquina were collected in Rio de Janeiro, Brazil, by Rita de Cássia Almeida Lafetá, FAETEC (Fundação de Apoio à Escola Técnica do Rio de Janeiro).A voucher specimen RFA40631 was deposited at the Herbário RFA, Departamento de Biologia, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro-RJ, Brazil.

Extraction and isolation
The fruits from S. pseudoquina were weighed (300 g) and pulverized in a laboratory blender with aqueous 5% acetic acid solution (1 L) at room temperature for 5 min.A 30 min ultrasonic assisted extraction was then performed.The solution was filtered over Celite ® and the residue was re-extracted with a new 1 L volume of acid solution.The aqueous acidic extracts were applied to an open column (50 × 6 cm) filled with 250 g of XAD-2 resin previously washed with pure methanol and then conditioned with 5% aqueous acetic acid solution.After applying the extract to the XAD-2 column, a column wash with 500 mL methanol:water (1:1) was done.An enriched SGA fraction was obtained with another column wash with pure methanol (1 L).The methanol fraction from XAD-2 was dried under vacuum at 45 ºC producing 11.4 g of residue.Around 4.85 g of the residue were dissolved in 250 mL of aqueous 1% acetic acid and concentrated NH 4 OH (28%) was added to force precipitation of the alkaloids.A crude mixture of SGA was obtained after centrifugation (1.9 g).Half of the crude mixture was subjected to column chromatography over silica (25 g) with mobile phase CH 2 Cl 2 :MeOH:2% aqueous NH 4 OH (650:350:50, v/v/v) and 6 mL fractions were collected.After repeating the column chromatography procedure, fractions 3 to 7 from both separations were pooled to yield, after evaporation, the alkaloidal fraction GA33 (404.6 mg).GA33 was further subjected to silica gel column chromatography, with mobile phase CH 2 Cl 2 :MeOH:2% aqueous NH 4 OH (785:200:15 v/v/v) and 6 mL collected fractions, to give GA33A (198.0 mg) from fractions 6 to 12.All silica gel chromatography were carried out at room temperature with flow of 12 mL min -1 .

Results and Discussion
The aqueous acidic extract of S. pseudoquina berries was subjected to chromatographic purification over a XAD-2 resin and repeated silica gel column chromatography, followed by semi preparative HPLC purification, to afford three new compounds (1, 2, and 3) (Figure 1).
Compound  1) displayed two superimposed three proton singlet resonances at d 0.65 (H-18 and H-19) assigned to two steroidal angular methyl groups and two three proton doublet signals at 1.13 (d, 3 J HH 6.9 Hz, H-21) and 0.83 (d, 3 J HH 6.6 Hz, H-27), characteristic of a steroidal alkaloid skeleton. 11The 13 C NMR spectrum of 1 exhibited 35 signals, consistent with a steroidal alkaloid plus one acetyl and one hexose residues.The edited heteronuclear single quantum coherence (HSQC) spectrum allowed the identification of five methyl groups (the one at d 21.47 assigned to acetyl group, C-29), twelve methylene and fourteen methine groups.The remaining four nonhydrogenated carbons were identified as: d 170.40 (ester carbonyl, C-28), 43.79 (C-13), 173.13 (C-22) and 35.71 (C-10).All these carbons were assigned with the help of a heteronuclear multiple bond correlation (HMBC) spectrum.The methyl groups correlations in the HMBC spectrum provided starting points to help assigning the remaining hydrogens and carbons in the steroidal backbone.The free rotation of these groups keeps the values for 2 J C,H and 3 J C,H around 4.5-6 Hz, allowing the detection of all expected methyl correlations. 12From the HMBC correlation map, the mutual 2 J C,H / 3 J C,H carbon/hydrogen correlations led to the identification of a sequence including 13 out of the 27 carbons in the aglycone as seen on Figure 2. The loss of 60.0211 plus 162.0528 mass units observed in the HRESIMS spectrum of 1 was taken as evidence that one acetyl and one hexose sugar residue were present.To confirm the identity of the sugar residue, a 1D total correlation spectroscopy (TOCSY) experiment was set up with increasing mixing times from 10 to 200 ms (array of 5) and selecting the signal attributed to the anomeric hydrogen at d Η 5.05 (d, J 7.7 Hz).This experiment allowed for the identification of the J connectivity array in the sugar moiety as similar to a β-anomeric glucopyranosyl residue as: d 5.05 (H-1'), 4.06 (H-2'), 4.05 (H-3'), 4.28 (H-4'), 4.31 (H-5') and 4.62/4.44(H-6'). 13,14The corresponding carbons were assigned by analyzing the edited HSQC spectrum.The observed HMBC cross correlation peak between the carbon signal at d 102.22 (C-1') and the proton signal at d 3.97 (H-3), indicated that the glucopyranosyl residue was linked to C-3 of the aglycone.This assignment was unambiguously confirmed by the observed ROE correlation between the H-1' anomeric proton and H-3.The rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiment also confirmed the equatorial β orientation of the oxygen at C-3, as well as the overall stereochemistry of the steroid skeleton.The structure of the epimino ring attached to carbon 20 was deduced as follows: the HMBC correlations of the C-27 (d 19.The results found so far led us to propose that compound 1 is a 22(N)-unsaturated 22,26-epimino-5αcholestane type steroidal alkaloid bearing an acetyl group at C-16 and a β-anomeric glucopyranosyl residue at C-3.The stereochemistry at C-20 was deduced from the value of the vicinal H-20/H-17 coupling constant (11.5 Hz), indicating an antiperiplanar orientation between those protons.Additionally, ROE cross peaks were observed between CH 3 -21/H-12β as well as between H-20/H-16 (Figure 3).These results are indicative of an S configuration at C-20.Regarding the 22,26-epimino-22(N)-ene ring, the 1D 1 H NMR of compound 1, dissolved in CD 3 OD, displayed a similar coupling pattern for protons 26α (d 2.95; 16.8 and 9.4 Hz) and 26β (d 3.52; 16.8, 4.8 and 1.5 Hz) as the one displayed by etioline, an alkaloid with 20S, 25S configuration. 15Thus, we may conclude that compound 1 is 3-O-(β-D-glucopyranosyl) (20S,25S)-22,26epimino-16α-acetyl-cholesta-22(N)-ene.The configuration at C-20 and C-25, for this class of alkaloids, is subject of discussion in literature and has been formerly determined by synthesis and, more recently, from scalar coupling and ROE measurements. 9,10,15,16Thus, compound 1 is a new 25-isosolafloridine type alkaloid bearing an acetyl group at C-16 and a β-anomeric glucopyranosyl residue at C-3.There is a previous report on the isolation of an alkaloid of similar skeleton but without the acetyl and glycosyl moieties, isolated from Solanum callium. 17 The NMR spectral data (Table 1) for compounds 2 and 3 are almost superimposable by comparison of their proton and carbon chemical shifts in rings A, B and C of the steroidal backbone and in the sugar residue.Additionally, comparison of the sugar residue chemical shift data for 2 and 3 confirms that a β-D-glucopyranosyl unit residue is common to the two compounds.For both 2 and 3 compounds 1D TOCSY experiments were set up with increasing mixing times from 10 to 200 ms and selecting the signal at d Η 4.37 (d, J 7.7 Hz).These experiments allowed for the identification of the J connectivity array in the sugar moiety as similar to a β-anomeric glucopyranosyl residue as: (2) d 4.37 (H-1'), 3.13 (H-2'), 3.33 (H-3'), 3.25 (H-4'), 3.24 (H-5') and 3.84/3.64(H-6') and for (3) d 4.38 (H-1'), 3.14 (H-2'), 3.34 (H-3'), 3.27 (H-4'), 3.27 (H-5') and 3.84/3.65(H-6'). 13,14The methyl groups correlations in the HMBC spectrum allowed the assignment of C-  were thus characterized as solaspiralidine-type alkaloids as the one isolated from S. spirale. 10Although the isolation of C-20 epimeric solaspiralidine alkaloids has already been described, e.g., from the bulbs of Fritillaria persica (Liliaceae), 18 this is the first report on the isolation of C-20 epimeric, 3-O-β-D-glucosyl, 16-acetyl solaspiralidine alkaloids.This rare structural alkaloid type is characterized by the double bond between C-23 and N in the fivemembered ring heterocycle and a carbonyl function at C-22.These compounds were originally interpreted as 22,26-epiminocholestanes. 3

Figure 1 .
Figure 1.Chemical structures of the alkaloids isolated from S. pseudoquina berries.