Synthesis and anti-tumour, immunomodulating activity of diosgenin and tigogenin conjugates.

A series of novel diosgenin (DSG) and tigogenin (TGG) derivatives with diosgenin or tigogenin steroid aglycons linked to levulinic and 3,4-dihydroxycinnamic acids, dipeptides and various amino acids by an ester bond at the C3-oxygen atom of the steroid skeleton has been synthesized. Diosgenyl esters have been prepared by an esterification reaction (DCC/DMAP) of diosgenin with the corresponding acids. All analogues have been evaluated in vitro for their antiproliferative profile against cancer cell lines (MCF-7, MDA-MB-231, PC-3) and human umbilical vein endothelial cells (HUVEC). Analogue2c (l-serine derivative of TGG), the best representative of the series showed IC50 of 1.5 µM (MCF-7), and induced apoptosis in MCF-7 by activating caspase-3/7. The immunomodulatory properties of six synthesized analogues have been determined by examining their effects on the expression of cytokine genes essential for the functioning of the human immune system (IL-1, IL-4, IL-10, IL-12 and TNF-α). Biological evaluation has revealed that new compounds 4c and 16a do not induce the expression of pro-inflammatory cytokines in THP-1 cells after the lipopolysaccharide (LPS) stimulation. They also stimulate the expression of anti-inflammatory IL-10 that acts stronger than diosgenin itself. An in silico ADME properties(absorption, distribution, metabolism, excretion) study was also performed to predict the pharmacokinetic profile of the synthesized compounds. To shed light on the molecular interactions between the synthesized compounds and the glucocorticoid receptor and the estrogen receptor, 2c, 4c and 16a compounds were docked into the active binding sites of these receptors. The in silico and in vitro data suggested that this new group of compounds might be considered as a promising scaffold for further modification of more potent and selective anticancer and immunomodulatory agents.

Access to 27-Nortomatidine and 27-Norsoladulcidine Derivatives.

Synthesis of (22 R)- and (22 S)-27-norspirosolane alkaloids from tigogenin, epismilagenin, and smilagenin is described. The alkaloids were prepared from readily available dinorcholanic lactones via their reaction with 4-chlorobutyllithium followed by substitution of chloride with azide and reductive N-cyclization under the Staudinger conditions.

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 of new sarsasapogenin derivatives with antiproliferative and apoptotic effects in MCF-7 cells.

Sarsasapogenin, a kind of mainly effective component of Anemarrhena asphodeloides Bunge, possesses good antitumor properties. Two series of new sarsasapogenin derivatives were synthesized and evaluated for their cytotoxicities against three human cancer cell lines (HepG2, A549, MCF-7) using the MTT assay. The structure-activity relationship revealed that the N, N-dimethylamino, pyrrolidinyl, and imidazolyl substituted at the C26 position could increase the antitumor efficacy of the 3-oxo sarsasapogenin series of compounds. Compound 4c with pyrrolidinyl substituted at the C26 position exhibited the greatest cytotoxic activity against MCF-7 cell line (IC50 = 10.66 µM), which was 4.3-fold more potent than sarsasapogenin. Action mechanism investigations showed that 4c could inhibit the colony formation and induce the apoptosis of MCF-7 cells. Further researches showed that a decrease in mitochondrial membrane potential and increases in the expression level of cleaved-PARP and the ratio of Bax/Bcl-2 were observed in MCF-7 cells after treatment with 4c, suggesting that the mitochondrial pathway was involved in the 4c-mediated apoptosis. These results show that compound 4c may serve as a lead for further optimization.

Synthesis of tigogenin MeON-Neoglycosides and their antitumor activity.

To discover new potent cytotoxic steroidal saponins, a series of tigogenin neoglycosides were synthesized via oxyamine neoglycosylation for the first time. The preliminary bioassays for their in vitro antitumor activities against five human cancer cell lines (A375, A-549, HCT-116, HepG2 and MCF-7) were conducted. The results revealed a sugar-dependent activity profile of their cytotoxicity, the glycoconjugation converted the non-active tigogenin to the most potential product Tg29 ((3R)-N-methoxyamino-tigogenin-ß-2-deoxy-d-galactoside) with IC50 value of 2.7µM and 4.6µM against HepG2 and MCF-7 cells respectively. And the 3R-tigogenin neoglycosides exhibited enhanced antitumor activity while the 3S-tigogenin almost showed no activity. Among the five cell lines, HepG2 and MCF-7 cells showed more sensitive cytotoxic responses to the products. Therefore, the neoglycosylation could be a promising strategy for the synthesis of antitumor steroidal saponins and it also proved the essential role of carbohydrate moiety of steroidal saponins in the biological activity.

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.

Synthesis and evaluation of 26-amino acid methyl ester substituted sarsasapogenin derivatives as neuroprotective agents for Alzheimer's disease.

Sarsasapogenin, extracted from Anemarrhena asphodeloides Bunge., has been reported to protect neurons from H2O2-induced damage. In the current study, four series of 26-amino acid methyl ester substituted sarsasapogenin derivatives (5a-5e, 5f-5j, 6a-6e and 7a-7e) were synthesized and tested for neuroprotective activity by evaluating their neuroprotective ratio against SH-SHY5Y cell lines. Studies showed that most of the target compounds displayed better neuroprotective effects than that of sarsasapogenin. Structure-activity relationship analysis suggested that 3-methoxy derivatives (5f-5j) were more potent than other series and the phenylalanine methyl ester moiety at C-26 was important for exhibiting apparent neuroprotective activity. It was worth noting that compound 5h exhibited optimal neuroprotective activity (102.2%) compared with sarsasapogenin (27.3%) and trolox (40.5%), and this encouraged us to investigate the cellular mechanism of 5h further. Our investigation revealed that 5h could attenuate H2O2-induced cell damage by inhibiting the expression of cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspase-3 as well as rescuing the downregulation of brain-derived neurotrophic factor (BDNF) and its tyrosine receptor kinase B (TrkB). Taken together, these results suggest that the representative compound 5h is a profound lead compound for further investigation and the sarsasapogenin skeleton could be a promising structural template for the development of new anti-Alzheimer drug candidates.

Synthesis of new sarsasapogenin derivatives with cytotoxicity and apoptosis-inducing activities in human breast cancer MCF-7 cells.

Based on the fact that Timosaponin A-III, a saponin isolated from the rhizome of Anemarrhena asphodeloides, is a promising bioactive lead compound in the treatment of cancer, structural modification at the C3 and C26 positions of sarsasapogenin has always been the focus of our structure-activity investigations. In this paper, we describe the synthesis of a range of new derivatives 5a-5o and the evaluation of their antitumor activities in a panel of six human cancer cell lines using the MTT assay in vitro. The results obtained showed that compounds 5h, 5i, and 5n exhibited significant cytotoxic activities against the six cell lines, being more potent than their parent compound sarsasapogenin. Furthermore, the p-fluorobenzyloxy series of compounds generally exhibited stronger cytotoxicities against all the tested cancer cells compared with the benzyloxy and p-methoxybenzyloxy series, and the substitution of pyrrolidinyl and piperazinyl groups at the C26 position was the preferred option for these compounds to display antitumor activities. Compound 5n exhibited excellent cytotoxic activity against MCF-7 cell line (IC50 = 2.95 µM), and was 16.7-fold more potent than sarsasapogenin. Further studies of the cellular mechanism of 5n showed that it arrested MCF-7 cells at the G2/M phase and induced apoptosis and necrosis. All these results show that it is important to carry out structural modification of sarsasapogenin to obtain some promising derivatives with marked antitumor activities, and the representative compound 5n is a lead compound for further research.

Synthesis and biological evaluation of novel sarsasapogenin derivatives as potential anti-tumor agents.

Based on the fact that timosaponin A-III (TA-III) exhibits potent cytotoxic effects and has been considered as a potential anti-tumor agent, a range of novel sarsasapogenin derivatives 1, 2a-2g, 3, 4, 5, 6a-6g have been synthesized by a simple and facile synthetic route. The in vitro cytotoxic activity of these synthetic compounds has been evaluated against ten human cancer cell lines. The pharmacological results showed that most of the sarsasapogenin derivatives displayed excellent selective cytotoxicity toward the cancer cell lines. An amino group at C-3 or C-26 position of the sapogenin had a profound influence on the cytotoxic activity. In particular, compound 6c exhibited significantly inhibitory activity against A375-S2 (IC50=0.56µM) and HT1080 (IC50=0.72µM) cells. However, introducing a bromo or morpholinyl substituent at the C-3 and C-26 position of the sapogenin generally rendered it inactive against the human cancer cell lines. This research provides a theoretical reference for the exploration of new anti-tumor drugs.

Efficient one-pot synthesis of tigogenin saponins and their antitumor activities.

An efficient synthesis of naturally occurring tigogenin triglycoside 1a and its three derivatives 1b-d bearing different carbohydrate moieties, as well as their antitumor activities, is described. Partially protected thiogalactosides bearing unprotected 2,4-OH or 4-OH groups were used to facilitate regioselective reactions for one-pot sequential multi-step glycosylation, which has significantly simplified the target molecule synthesis. The synthetic saponins 1a-d exhibited much higher anti-tumor activities than the positive control cisplatin against the human epithelial cervical cancer cell (HeLa) as evaluated by CCK-8 assay.

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.

New insights into the structure-cytotoxicity relationship of spirostan saponins and related glycosides.

A variety of spirostan saponins and related glycosides were synthesized and evaluated for their cytotoxicity against the human myeloid leukemia cell line (HL-60). A linear glycosylation strategy allowed for accessing a variety of functionalization patterns at both the spirostanic and the saccharide moieties, which provides new information regarding the structure-cytotoxicity relationship of this family of steroidal glycosides. Intriguing results were achieved with respect to hecogenyl and 5α-hydroxy-laxogenyl ß-chacotriosides, turning out to be the former very cytotoxic and the latter no cytotoxic at all. Importantly, the partially pivaloylated ß-d-glucosides of 5α-hydroxy-laxogenin were the most potent cytotoxic compounds among all tested glycosides. This comprises the first report on acylated spirostanyl glucosides displaying significant cytotoxicity, and therefore, it opens up new opportunities toward the development of saponin analogues as anticancer agents.

Synthesis and antifungal activity of functionalized 2,3-spirostane isomers.

Invasive fungal infections are a major complication for individuals with compromised immune systems. One of the most significant challenges in the treatment of invasive fungal infections is the increased resistance of many organisms to widely used antifungals, making the development of novel antifungal agents essential. Many naturally occurring products have been found to be effective antimicrobial agents. In particular, saponins with spirostane glycosidic moieties-isolated from plant or marine species-have been shown to possess a range of antimicrobial properties. In this report, we outline a novel approach to the synthesis of a number of functionalized spirostane molecules that can be further used as building blocks for novel spirostane-linked glycosides and present results from the in vitro screenings of the antifungal potential of each derivative against four fungal species, including Candida albicans, Cryptococcus neoformans, Candida glabrata, and the filamentous fungus Aspergillus fumigatus.

Synthesis and anti-glioma activity of 25(R)-spirostan-3beta,5alpha,6beta,19-tetrol.

Malignant gliomas are common and aggressive brain tumours in adults. The rapid proliferation and diffuse brain migration are the main obstacles to successful treatment. Here, we show 25(R)-spirostan-3beta,5alpha,6beta,19-tetrol, a polyhydroxy steroid, is capable of suppressing proliferation and migration of C6 malignant glioma cells in a concentration-dependent manner. The compound 25(R)-spirostan-3beta,5alpha,6beta,19-tetrol was synthesised by seven steps starting from diosgenin in 8.55% overall yield. The structures of the synthetic compounds were characterised by infrared (IR), (1)H nuclear magnetic resonance (NMR), (13)C NMR spectra and EA.

Synthesis of "glycospirostanes" via ring-closing metathesis.

Syntheses of "glycospirostanes" from 3beta-hydroxy-23,24-dinor-5alpha-cholano-22,16-lactone and 3alpha-hydroxy-23,24-dinor-5beta-cholano-22,16-lactone were performed. The key step of these syntheses was ring-closing metathesis of the corresponding C,O-diallyl derivative. Further elaboration of the six-membered ring F consisted of allylic hydroxylation with SeO(2) followed by OsO(4) dihydroxylation of the C24-C25 double bond. The obtained final products proved to be simultaneously O- and C-l-arabinopyranosides.

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.

Synthesis of "glycospirostanes"--steroid sapogenins with a sugar-like ring F.

The synthesis of two "glycospirostanes" from 23-oxotigogenin acetate is described. (23S,24S,25R)-5alpha-Spirostane-3beta,23,24,25-tetraol was obtained by dehydrogenation followed by stereoselective reduction of the 23-oxo group and OsO(4) dihydroxylation of the C24-C25 double bond. Allylic hydroxylation with SeO(2) of 3beta-acetoxy-5alpha-spirost-23-ene obtained from 23-oxotigogenin acetate followed by OsO(4) dihydroxylation of the C23-C24 double bond afforded (23R,24S,25R)-5alpha-spirostane-3beta,23,24,25-tetraol.

Facile synthesis of 12-carboxamido-11-spirostenes via palladium-catalyzed carbonylation reactions.

12-Carboxamido- and 12-carboxyl-11-spirostenes were synthesized from the corresponding 12-iodo-11-ene derivative in palladium-catalyzed carbonylation reactions under mild reaction conditions. The synthesis of the iodo-alkene substrate is based on the transformation of the 12-keto derivative (hecogenin) to hydrazone, which was treated with iodine in the presence of a base (1,1,3,3-tetramethyl guanidine). While various 12-carboxamides were synthesized in moderate to high yields by using simple alkyl/arylamines or amino acid methylesters as N-nucleophiles, low yields can be achieved with alcohols as O-nucleophiles. The homogeneous carbonylation reactions tolerate the 3-hydroxy substituent and the spiroacetal moiety.

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.