Characterization of 3-aminospirostane alkaloids from roots of Solanum paniculatum L. with hepatoprotective activity.

RATIONALE: Solanum paniculatum L., popularly known as jurubeba, has traditionally been used in Brazilian folk medicine for liver diseases. However, there is a lack of knowledge about the chemical characterization of 3-aminospirostane alkaloids, an important class related to pharmacological activities. This work aimed to characterize the alkaloids using liquid chromatography with tandem mass spectrometry (LC/MS/MS) supported by molecular networking and theoretical calculations as well as to evaluate the contribution to hepatoprotective activity. METHODS: S. paniculatum roots were collected and macerated with MeOH/H2 O (8:2) obtaining the crude extract (SP-CE). From this, partition using EtOAc with pH variation yielded the alkaloidic fraction (SP-AF). Both were evaluated in an acute liver injury model (100 and 200 mg/kg), after intraperitoneal administration of carbon tetrachloride (CCl4 ) in mice. AST (aspartate transaminase) and ALT (alanine transaminase) serum levels were investigated, as well as the histopathological characteristics. The SP-CE and SP-AF were analyzed by LC/MS/MS, using quadrupole/time-of-flight and ion-trap systems. The alkaloids annotated by the GNPS molecular network had their structures defined using gas-phase ionization and fragmentation reaction supported by theoretical calculations. RESULTS: The SP-CE and SP-AF decreased the ALT serum levels compared with the negative control. The group treated with the SP-CE (at the highest dose) demonstrated a significant decrease of ALT. Hepatic cell degeneration decrease was observed mainly at the highest dose of the treatment. Detailed electrospray ionization MS/MS data allowed us to identify alkaloids not previously reported, to propose their gas-phase reactions and to redefine the initial open ring fragmentation mechanism of the steroidal alkaloids with the jurubidine moiety. CONCLUSIONS: The results allowed us to identify seven steroidal alkaloids from jurubeba and redefine the initial mechanism of fragmentation. A significant hepatoprotective effect was also demonstrated, corroborating its traditional use.

Studies on the reactivity of 23E-benzylidene spirostanes.

A study of the reactivity of 25R and 25S 23E-benzylidene spirostanes that includes epoxidation, catalytic hydrogenation as well as Lewis or Brønsted acid-catalyzed rearrangements is described. Exhaustive NMR characterization of the obtained compounds is presented. Additionally the structures of some of the obtained compounds were confirmed by single crystal X-Ray Diffraction studies.

Synthesis of dimeric spirostanols linked through a 1,4-dimethylidenebenzene moiety by double BF3·Et2O-catalyzed aldol condensation of steroid sapogenins and terephtalaldehyde.

BF3·Et2O-catalyzed double aldol condensation between acetylated steroid sapogenins and terephtalaldehyde led to acetylated dimeric spirostanols linked through a 1,4-dimethylidenebenzene moiety in moderate to good yields. The E configurations of the introduced double bonds were corroborated by NOE experiments. Saponification of the dimeric steroids led to the corresponding dimeric spirostanols.

Synthesis and in vitro anticancer activity of 23(23')E-benzylidenespirostanols derived from steroid sapogenins.

Benzylidenespirostanols were prepared by two-step synthesis including BF3·Et2O-catalyzed aldol condensation of several acetylated steroid sapogenins with benzaldehyde followed by saponification. The obtained compounds showed moderate cytotoxicity against three cancer cell lines (T-lymphoblastic leukemia cell line CEM, breast carcinoma cell line MCF7 and cervical carcinoma cell line HeLa) and normal human fibroblasts (BJ). The most active of the five tested substances was 3c (lowest IC50 for MCF7 cells 19.9±0.1µM) without any selectivity towards human cancer and normal cells, respectively.

Spirostanol glucosides from the leaves of Cestrum laevigatum L.

Two new steroidal saponins, (25R)-spirost-5-ene-3ß,26ß-diol 3-O-α-L-rhamnopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 4)-[(1 → 2)-α-L-rhamnopyranosyl]-ß-D-glucopyranoside (1) and (25R)-spirost-6-ene-3ß,5ß-diol 3-O-α-L-rhamnopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 4)-[(1 → 2)-α-L-rhamnopyranosyl]-ß-D-glucopyranoside (2), along with the known diosgenin 3-O-α-L-rhamnopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 4)-ß-D-glucopyranoside (3), chonglouoside SL-5 (4) and Paris saponin Pb (5) were isolated from the leaves of Cestrum laevigatum. The structures of the compounds were determined using spectroscopic analyses including HRESI-MS, 1D and 2D NMR data, followed by comparison with data from the literature. Among them, two are particularly unique, compound 1 is the first (6)Δ-spirostanol saponin and compound 2 has an unusual C-26 hydroxyl in the (5)Δ-spirostanol skeleton. Antifungal testing showed a potent activity to formosanin C against Candida albicans and Candida parapsilosis. Evaluation of the cytotoxic activity indicated that compound 1 has a moderate activity against HL-60 and SF-295 cell lines, while compound 2 were active only against HL-60.

Diacetoxyiodobenzene-mediated synthesis of unnatural furospirostane sapogenins derived from diosgenin and tigogenin.

Two unnatural steroid sapogenins bearing a furospirostane side chain were prepared starting from the readily available spirostane sapogenins, tigogenin and diosgenin following a synthetic protocol that included: (i) introduction of a carbonyl group at position C-23, (ii) diacetoxyiodobenzene-induced F-ring contraction and (iii) LiAlH4 reduction of the newly emerged methoxycarbonyl moiety. The structures of the new compounds were corroborated by NMR and X-ray studies.

Mechanistic insights on the reactivity of furospirostanes with the 16ß,22:22,25-diepoxy-23-acetoxymethyl-24-methyl side chain.

F-ring opening in spirostanes with the 16ß,22:22,25-diepoxy-23-acetoxymethyl-24-methyl side chain produces a Δ(22)-intermediate with an allylic acetoxy group. For this reason the reactivity profile of these compounds deviates from that observed in other naturally occurring or synthetic spirostanes and furospirostanes.

Multicomponent ligation of steroids: creating diversity at the linkage moiety of bis-spirostanic conjugates by Ugi reactions.

The diversity-oriented synthesis of novel bis-spirostanic conjugates utilizing two different Ugi four-component reactions (Ugi-4CR) is described. Spirostanic steroids were functionalized with Ugi-reactive groups, that is, amines, isocyanides, and carboxylic acids, and next were subjected to multicomponent ligation approaches leading to bis-steroidal conjugates featuring pseudo-peptidic and heterocyclic linkage moieties. Both the classic Ugi-4CR and its hydrazoic acid variant were implemented, proving good efficiency for the ligation of isocyanosteroids to spirostanic acids and equatorial amines. Axially oriented amines showed poorer results, although model studies proved that chemical efficiency could be significantly improved while increasing reaction times. Overall, the method comprises the rapid generation of molecular diversity at the bis-steroid linkage moiety and, consequently, shows promise toward the production of combinatorial libraries of bis-spirostanes for biological screening.

Carbohydrate-steroid conjugation by Ugi reaction: one-pot synthesis of triple sugar/pseudo-peptide/spirostane hybrids.

The one-pot synthesis of novel molecular chimeras incorporating sugar, pseudo-peptide, and steroidal moieties is described. For this, a new carbohydrate-steroid conjugation approach based on the Ugi four-component reaction was implemented for the ligation of glucose and chacotriose to spirostanic steroids. The approach proved wide substrate scope, as both mono and oligosaccharides functionalized with amino, carboxy, and isocyano groups were conjugated to steroidal substrates in an efficient, multicomponent manner. Two alternative strategies based on the hydrazoic acid variant of the Ugi reaction were employed for the synthesis of tetrazole-based chacotriose-diosgenin conjugates resembling naturally occurring spirostan saponins. This is the first time that triple sugar/pseudo-peptide/steroid hybrids are produced, thus opening up an avenue of opportunities for applications in drug discovery and biological chemistry.

Effect of C-ring modifications on the cytotoxicity of spirostan saponins and related glycosides.

Twelve C-ring modified spirostanyl glycosides were synthesized and evaluated for their cytotoxicity against the human myeloid leukemia cell line (HL-60). With the aim of assessing the influence of the hydrophobic character, the conformational flexibility and the stereochemistry of the C-ring functionalities on the cytotoxic activity, a variety of spirostanic aglycones incorporating methylene, methoxyl, α,ß-unsaturated ketone and lactone groups were subjected to a linear glycosylation strategy leading to glycosides derived from the 3,6-dipivaloylated ß-D-glucoside and the ß-chacotrioside moieties. The 3,6-dipivaloylated spirostanyl ß-D-glucosides showed moderate to good cytotoxic activity against HL-60, but no significant cytotoxicity against benign blood cells. However, the cytotoxicity of spirostanyl ß-chacotriosides was highly dependent on the nature of the C-ring functional groups of the steroidal aglycones. Actually, the chacotrioside-based saponins either with no functionality or bearing a hydrophobic methylene group at C-12 were the most cytotoxic ones against both HL-60 and benign blood cells. On the other hand, the incorporation of very polar functionalities and the opening of the ring C with the consequent loss of rigidity led to a significant drop in the cytotoxicity against HL-60. These results confirm that spirostanyl ß-chacotriosides including very lipophilic aglycones are the most cytotoxic ones among their congeners.

Regioselective cleavage of 22-oxo-23-spiroketals. Novel cholestanic frameworks with pyranone and cyclopentenone E rings on the side chain.

The regioselective opening of the F ring of 22-oxo-23-spiroketals using a saturated solution of HCl in acetic anhydride yielded novel cholestanic frameworks with pyranone or cyclopentenone E rings. The structures of the new derivatives of sarsasapogenin, diosgenin and hecogenin thus obtained were established using one and two dimensional (1)H, (13)C experiments (DEPT, COSY, HETCOR, HMBC, ROESY, and NOESY). The X-ray analysis for compound 11b confirmed the 23R configuration for the new stereogenic center.

Haemolytic activity and immunological adjuvant effect of a new steroidal saponin from Allium ampeloprasum var. porrum.

A new steroidal saponin was isolated from the bulbs of Allium ampeloprasum L. var. porrum. On the basis of chemical evidence, comprehensive spectroscopic analyses, and comparison with known compounds, its structure was established as (3ß,5α,6ß,25R)-3-{(O-ß-D-glucopyranosyl-(1→3)-ß-D-glucopyranosyl-(1→2)-O-[O-ß-D-glucopyranosyl-(1→3)]-O-ß-D-glucopyranosyl-(1→4)-ß-D-galactopyranosyl)oxy}-6-hydroxyspirostan-2-one (1). Results of the present study indicated that 1 exhibited haemolytic activity in the in vitro assays, and immunological adjuvant activity on the cellular immune response against ovalbumin antigen.

Rapid conversion of spirostans into furostan skeletons at room temperature.

We report a facile protocol to obtain 22-substituted furostans and pseudosapogenins in high yields from (25R)- and (25S)-sapogenins. This method involves the treatment of the sapogenin with acetic-trifluoroacetic mixed anhydride and BF(3)·OEt(2) at room temperature, followed by the addition of a nucleophile (H(2)O, MeOH or KSeCN). In the case of 22-hydroxyfurostans, they can be transformed to pseudosapogenins by treatment with p-toluensulfonic acid.

On the reactivity of 23-methoxycarbonyl furospirostanes.

Brønsted and Lewis acid-catalysed reactions of the 23-methoxycarbonyl furospirostanic side chain are described. While bromination, deuteration and BF(3)·Et(2)O/AcOH treatment involve regioselective F-ring opening with exclusive participation of Δ(22)-furostenic intermediates, BF(3)·Et(2)O/Ac(2)O treatment leads to irreversible E- or F-ring cleavage.

Deacylation reactions of 20-acetyl dinorcholanic lactones and 20,23-diacetyl furost-22-enes.

We report the deacylation of (20R)-20-acetyl-23,24-dinorcholanic lactones by hydrazine hydrate, under microwave irradiation in high yields. The elimination of the 20-acetyl group proceeded with retention of configuration which contrast with other proved deacylation methods that yield a mixture of diastereoisomers. In this way, unnatural (20R)-23,24-dinorcholanic lactones can be produced rapidly on a large scale. Both (20R)- and (20S)-lactones were prepared starting from diosgenin, hecogenin and sarsasapogenin, in 72-80% overall yields.

Revisiting 23-iodospirostanes. New facts and full characterization.

In addition to a previous report, the reaction of tigogenin acetate with ICl in refluxing CHCl(3) produced the hitherto unknown 23R-iodotigogenin acetate, bearing an axial iodine atom at C-23 and its already reported 23S-epimer. The same treatment of sarsasapogenin acetate led to a single diasteromer characterized as 23S-iodosarsasapogenin acetate. A full characterization of the obtained compound including (1)H, (13)C NMR, MS and X-ray diffraction is provided.

A new steroidal saponin from Agave shrevei.

A new steroidal saponin was isolated from the leaves of Agave shrevei. Its structure was established as 3-[O-beta-D-glucopyranosyl-(1-->2)-O-[O-beta-D-glucopyranosyl-(1-->4)-O-[O-beta-D-glucopyranosyl-(1-->6)]-O-beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranosyl)-oxy]-(3beta,5alpha,25R)-spirostane. The structural identification was performed using detailed analyses of 1H- and 13C-NMR spectra including 2D NMR spectroscopic techniques (COSY, HETCOR, HMBC, and HMQC) and chemical conversions. The haemolytic activity of the steroidal saponin was evaluated using an in vitro assay.

Novel 5beta-hydroxyspirostan-6-ones ecdysteroid antagonists: synthesis and biological testing.

Eight new 5beta-hydroxy-spirostan-6-ones bearing hydroxy and amino functions in the A ring, i.e., 3beta-OH, 3alpha-OH, 2beta,3beta-OH, 2alpha,3beta-OH, 3beta-NH2, 2alpha-NH2-3beta-OH, 2beta-NH2-3beta-OH, and 2beta-OH-3beta-NH2, were efficiently synthesized, and their antiecdysteroid activities were evaluated on the metamorphosis bioassay of mosquito Aedes aegypti. To our knowledge, these new steroids represent the first 5beta-hydroxy-spirostanes which have been tested for antiecdysteroid activity in mosquitoes. The higher antagonistic effect was found for compounds bearing the 3beta-hydroxy and 2beta,3beta-dihydroxy functionality, which show promise as environmental friendly insecticides.

New bisfuran derivative from sarsasapogenin. An X-ray and NMR analysis.

The new bisfuran derivative, (22S,23S)-22,23-dihydroxy-23,26-epoxyfurostane, was obtained from the known oxidation of sarsasapogenin acetate with NaNO2/BF3 in 5% aqueous acetic acid. The structure of was established using one and two-dimensional 1H, 13C experiments (DEPT, COSY, HETCOR and HMBC) and the configurations at the newly formed stereogenic centers were established as 22S,23S by an X-ray diffraction analysis. Addition of TiCl4 to bisfuran 5 confirmed that this compound is an intermediate in the rearrangement to 22-oxo-23-spiroketals since it was transformed quantitatively into the latter product. The 23-nitroimino intermediate 2 was isolated from the same reaction and its structure established also by an X-ray diffraction analysis; this compound was further transformed into the 23-nitramine 7 which could find application in functionalization of position 24.

Polyhydroxylated spirostanol saponins from the tubers of Dioscorea polygonoides.

Three new polyhydroxylated spirostanol saponins (1-3) were isolated from the tubers of Dioscorea polygonoides. The structures of these new compounds were determined on the basis of extensive spectroscopic analysis and the results of acid or enzymatic hydrolysis as (23S,24R,25S)-23,24-dihydroxyspirost-5-en-3beta-yl O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranoside (1), (23S,25R)-12alpha,17alpha,23-trihydroxyspirost-5-en-3beta-yl O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranoside (2), and (23S,25R)-14alpha,17alpha,23-trihydroxyspirost-5-en-3beta-yl O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranoside (3), respectively.