Steroidal glycosides from Ornithogalum thyrsoides bulbs and their cytotoxicity toward HL-60 human promyelocytic leukemia cells and SBC-3 human small-cell lung cancer cells.

Ornithogalum thyrsoides Jacq belongs to the Asparagaceae family and is cultivated for ornamental purposes. The authors have previously reported several cholestane- and spirostan-type steroidal glycosides from O. thyrsoides. Conventional TLC analysis of the methanolic bulb extract of O. thyrsoides suggested the presence of unprecedented compounds; therefore, a detailed phytochemical investigation of the extract was performed and 35 steroidal glycosides (1-35), including 21 previously undescribed ones (1-21) were collected. The structures of 1-21 were determined mainly by analyses of their 1H and 13C NMR spectra with the aid of two-dimensional NMR spectroscopy. The isolated compounds were classified into three distinct groups: furostan-type (1, 2, 8-12, and 22), spirostan-type (3-7 and 23-26), and cholestane-type (13-21 and 27-35). Although the C/D-ring junction of the steroidal skeleton is typically trans-oriented, except for some cardiotonic and pregnane-type steroidal derivatives, 7 possess a cis C/D-ring junction. This is the first reported instance of such a configuration in spirostan-type steroidal derivatives, marking it as a finding of significant interest. Compounds 1-35 were evaluated for cytotoxicity against HL-60 human promyelocytic leukemia cells and SBC-3 human small-cell lung cancer cells. Compounds 3-6, 9, 17-21, 23-25, and 30-35 demonstrated cytotoxicity in a dose-dependent manner with IC50 values ranging from 0.000086 to 18 µM and from 0.00014 to 37 µM toward HL-60 and SBC-3 cells, respectively. Compound 19, which is obtained in a good yield and shows relatively potent cytotoxicity among the undescribed compounds, induces apoptosis in HL-60 cells, accompanied by arresting the cell cycle of HL-60 cells at the G2/M phase. In contrast, 19 causes oxidative stress-associated necrosis in SBC-3 cells. The cytotoxic mechanism of 19 is different between HL-60 and SBC-3 cells.

From Marine Metabolites to the Drugs of the Future: Squalamine, Trodusquemine, Their Steroid and Triterpene Analogues.

This review comprehensively describes the recent advances in the synthesis and pharmacological evaluation of steroid polyamines squalamine, trodusquemine, ceragenins, claramine, and their diverse analogs and derivatives, with a special focus on their complete synthesis from cholic acids, as well as an antibacterial and antiviral, neuroprotective, antiangiogenic, antitumor, antiobesity and weight-loss activity, antiatherogenic, regenerative, and anxiolytic properties. Trodusquemine is the most-studied small-molecule allosteric PTP1B inhibitor. The discovery of squalamine as the first representative of a previously unknown class of natural antibiotics of animal origin stimulated extensive research of terpenoids (especially triterpenoids) comprising polyamine fragments. During the last decade, this new class of biologically active semisynthetic natural product derivatives demonstrated the possibility to form supramolecular networks, which opens up many possibilities for the use of such structures for drug delivery systems in serum or other body fluids.

A Total of Eight Novel Steroidal Glycosides Based on Spirostan, Furostan, Pseudofurostan, and Cholestane from the Leaves of Cestrum newellii.

Previously, various steroidal glycosides were reported from plants of Cestrum species. However, phytochemical investigation has not been conducted on Cestrum newellii. A systematic phytochemical investigation of the leaves of C. newellii resulted in the isolation of eight novel steroidal glycosides (1-8), which were classified into three spirostanol glycosides (1-3), two furostanol glycosides (4 and 5), two pseudofurostanol glycosides (6 and 7), and one cholestane glycoside (8). In addition, three known cholestane glycosides (9-11) were isolated and identified. The structures of the new compounds were determined based on spectroscopic data and chemical transformations. Compounds 1 and 2 are spirostanol glycosides having hydroxy groups at C-2, C-3, C-12, and C-24 of the aglycone moiety. Although C. newellii is known to be a poisonous plant, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay exhibited that none of the isolated compounds were cytotoxic to HL-60 human promyelocytic leukemia cells.

3ß, 6ß-dichloro-5-hydroxy-5α-cholestane facilitates neuronal development through modulating TrkA signaling regulated proteins in primary hippocampal neuron.

Potentiating neuritogenesis through pharmacological intervention might hold therapeutic promise in neurodegenerative disorders and acute brain injury. Here, we investigated the novel neuritogenic potentials of a steroidal chlorohydrin, 3ß, 6ß-dichloro-5-hydroxy-5α-cholestane (hereafter, SCH) and the change in cellular proteome to gain insight into the underlying mechanism of its neurotrophic activity in hippocampal neurons. Morphometric analysis showed that SCH promoted early neuronal differentiation, dendritic arborization and axonal maturation. Proteomic and bioinformatic analysis revealed that SCH induced upregulation of several proteins, including those associated with neuronal differentiation and development. Immunocytochemical data further indicates that SCH-treated neurons showed upregulation of Hnrnpa2b1 and Map1b, validating their proteomic profiles. In addition, a protein-protein interaction network analysis identified TrkA as a potential target connecting most of the upregulated proteins. The neurite outgrowth effect of SCH was suppressed by TrkA inhibitor, GW441756, verifying TrkA-dependent activity of SCH, which further supports the connection of TrkA with the upregulated proteins. Also, the computational analysis revealed that SCH interacts with the NGF-binding domain of TrkA through Phe327 and Asn355. Collectively, our findings provide evidence that SCH promotes neuronal development via upregulating TrkA-signaling proteins and suggest that SCH could be a promising therapeutic agent in the prevention and treatment of neurodegenerative disorders.

Structure and bioactivity of cholestane glycosides from the bulbs of Ornithogalum saundersiae Baker.

Eight undescribed cholestane glycosides named osaundersioside A-H, along with three previously known compounds named osaundersioside I-K were isolated from Ornithogalum saundersiae Baker bulbs (Asparagaceae). Their structures were elucidated by extensive spectroscopic analysis and chemical methods. All isolates were evaluated for their cytotoxic activity and inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production. Osaundersioside C was thus determined to exhibit specific cytotoxicity towards MCF-7 cell line with an IC50 value of 0.20 µM, Osaundersioside H exhibited inhibitory effect on NO production in macrophages at the concentration of 10-5 M, with inhibition rate of 56.81%.

22-Oxocholestane oximes as potential anti-inflammatory drug candidates.

22-Oxocholestanes bearing the oxime functionality in the side chain have been synthesized from diosgenin and evaluated in vivo as anti-inflammatory agents in an acute inflammation mouse ear model, against the commercial glucocorticoid dexamethasone. The final compounds were all regioselectively obtained with an E configuration at the oxime double bond. The title compounds reduced ear-induced inflammation and edema. The most active oximes repressed the expression of proinflammatory genes TNF-α, COX-2, and IL-6; including macrophage migration inhibitory factor. Overall, our data suggest that 22-oxocholestane oximes exert a strong in vivo anti-inflammatory activity.

Synthesis and Evaluation of Novel Cholestanoheterocyclic Steroids as Anticancer Agents.

Modification of steroid molecules by introducing heterocyclic ring into the core structure of steroids has been utilized as an attractive approach for either cancer prognosis or diagnosis. Several new cholestanoheterocyclic steroids were synthesized, and analytical and spectral data proved the validity of the novel synthesized steroid derivatives. The cytotoxicity of synthesized compounds 3, 4, 5, 7, 9, 10, 13, 15b, and 16b was evaluated using human colorectal cancer HCT 116 and Caco-2, cervical cancer HeLa, hepatoma HepG2, and breast cancer MCF7 cell lines. Intriguingly, compound 13 has the highest cytotoxic effect when applied on the majority of cancer cells. In conclusion, compound 13 may be considered as a promising anticancer candidate against all cancer cell lines, because it recorded the lowest IC50 of the majority of the cancer cell lines used. Furthermore, a molecular docking study was employed to determine the binding modes against aromatase cytochrome P450 (CYP19), cyclin-dependent kinase 2 (CDK2), and B-cell lymphoma (BCL-2) proteins, which are major proteins involved in the pathogenesis of cancer. Molecular docking analyses revealed that compounds 13, 3, and 5 (free energy of binding = - 9.2, - 9.1, and - 9.0 kcal/mol, respectively) were the best docked ligand against aromatase CYP19; compounds 16b, 3, 9, and 10 (free energy of binding = - 9.6, - 9.3, and - 9.2 kcal/mol, respectively) were the best docked ligand against CDK2, while compounds 15b, 16b, and 13 (free energy of binding = - 9.1, - 9.0, and- 8.7 kcal/mol, respectively) were the best docked ligand against BCL2. In conclusion, compounds 3, 13, and 16b were the most promising compounds with the lowest IC50s against most of the tested cancer cell lines, and they displayed the lowest binding energies, critical hydrogen bonds, and hydrophobic interactions with the three molecular targets compared to other tested compounds.

Cholestane glycosides from Ornithogalum saundersiae bulbs and the induction of apoptosis in HL-60 cells by OSW-1 through a mitochondrial-independent signaling pathway.

A search for cytotoxic cholestane glycosides from Ornithogalum saundersiae bulbs resulted in the isolation of three new OSW-1 analogues (1-3), a new cholestane bisdesmoside (4), a 5ß-cholestane diglycoside (5), and four new 24(23 → 22)-abeo-cholestane glycosides (6-9), together with 11 known cholestane glycosides (10-20), including OSW-1 (11). The structures of 1-9 were determined based on conventional spectroscopic analysis and chemical evidence. As expected, based on previous data, 1-3 exhibited potent cytotoxic activity against HL-60 human promyelocytic leukemia cells and A549 human lung adenocarcinoma cells. Furthermore, the ability of OSW-1 to induce apoptosis in HL-60 cells was examined. Aggregation of nuclear chromatin, accumulation of the sub-G1 cells, DNA fragmentation, and caspase-3 activation were assessed in HL-60 cells treated with OSW-1, providing evidence for OSW-1-induced apoptosis in HL-60 cells. No mitochondrial membrane potential or release of cytochrome c into the cytoplasm were observed in the OSW-1-treated apoptotic HL-60 cells, indicating that a mitochondria-independent signaling pathway is involved in apoptotic cell death.

Structural Characterization of Cholestane Rhamnosides from Ornithogalum saundersiae Bulbs and Their Cytotoxic Activity against Cultured Tumor Cells.

Previous phytochemical studies of the bulbs of Ornithogalum saundersiae, an ornamental perennial plant native to South Africa, resulted in the isolation of 29 new cholestane glycosides, some of which were structurally unique and showed potent cytotoxic activity against cultured tumor cell lines. Therefore, we aimed to perform further phytochemical examinations of methanolic extracts obtained from Ornithogalum saundersiae bulbs, isolating 12 new cholestane rhamnosides (1-12) and seven known compounds (13-19). The structures of the new compounds (1-12) were identified via NMR-based structural characterization methods, and through a sequence of chemical transformations followed by spectroscopic and chromatographic analysis. The cytotoxic activity of the isolated compounds (1-19) and the derivatives (1a and 6a) against HL-60 human promyelocytic leukemia cells and A549 human lung adenocarcinoma cells was evaluated. Compounds 10-12, 16, and 17 showed cytotoxicity against both HL-60 and A549 cells. Compound 11 showed potent cytotoxicity with an IC50 value of 0.16 µM against HL-60 cells and induced apoptotic cell death via a mitochondrion-independent pathway.

Homo-aro-cholestane, furostane and spirostane saponins from the tubers of Ophiopogon japonicus.

Phytochemical investigation of the tubers of Ophiopogon japonicus led to the isolation of five previously undescribed steroidal saponins, ophiojaponins A-E, together with twelve known ones. The structures of these isolated compounds were elucidated by detailed spectroscopic analyses and chemical methods. Ophiojaponins A-C are rare naturally occurring C29 steroidal glycosides possessing a homo-cholestane skeleton with an aromatized ring E. Ruscogenin 1-O-α-L-rhamnopyranosyl-(1 â†’ 2)-4-O-sulfo-ß-D-fucopyranosido-3-O-ß-D-glucopyranoside was isolated as single component and its full spectroscopic data was reported for the first time. The isolated steroidal saponins were evaluated for their cytotoxicities against two human tumor cell lines MG-63 and SNU387. Among them, five known spirostane-type glycosides showed cytotoxic activity against both MG-63 and SNU387 cell lines with IC50 values ranging from 0.76 to 27.0 µM.

Structure activity relationship studies on cytotoxicity and the effects on steroid receptors of AB-functionalized cholestanes.

Structure-activity relationship analysis and profiling of a library of AB-functionalized cholestane derivatives closely related to brassinosteroids (BRs) were performed to examine their antiproliferative activities and activities on steroid hormone receptors. Some of the compounds were found to have strong cytotoxic activity in several human normal and cancer cell lines. The presence of a 3-hydroxy or 3-oxo group and 2,3-vicinal diol or 3,4-vicinal diol moiety were found to be necessary for optimum biological activity, as well as a six-membered B ring. According to the profiling of all steroid receptors in both agonist and antagonist mode, the majority of the cholestanes were weakly active or inactive compared to the natural ligands. Estrogenic activity was detected for two compounds, two compounds possessed antagonistic properties on estrogen receptors and seven compounds showed agonistic activity. Two active cholestane derivatives were shown to strongly influence cell viability, proliferation, cell cycle distribution, apoptosis and molecular pathways responsible for these processes in hormone-sensitive/insensitive (MCF7/MDA-MB-468) breast cancer cell lines.

In vitro cytotoxcity and interaction of new steroidal oxadiazinanones with calf thymus DNA using molecular docking, gel electrophoresis and spectroscopic techniques.

Herein we report synthesis of new steroidal oxadiazinanones from steroidal ketones. After characterization by spectral and analytical data, the interaction studies of compounds (4-6) with DNA were carried out by UV-vis, fluorescence spectroscopy and gel electrophoresis. The compounds bind to DNA preferentially through electrostatic and hydrophobic interactions with Kb; 1.8×10(4) M(-1), 2.2×10(4) M(-1) and 2.6×10(4) M(-1), respectively, indicating the higher binding affinity of compound 6 towards DNA. Gel electrophoresis showed the concentration dependent cleavage activity of compound 6 alone or in presence of Cu (II) causes the nicking of supercoiled pBR322 and it seems to follow the mechanistic pathway involving generation of hydroxyl radicals that are responsible for initiating DNA strand scission. Molecular simulations suggest that compounds binds through minor groove of DNA. MTT assay depicted promising anticancer activity of compound 5 and 6 particularly against HL-60 and MCF-7. The apoptotic degradation of DNA was analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining (comet assay). The results revealed that compound 6 has better prospectus to act as cancer chemotherapeutic candidate which warrants further in vivo anticancer investigations.

Functional properties of Claramine: a novel PTP1B inhibitor and insulin-mimetic compound.

Protein tyrosine phosphatase 1B (PTP1B) inhibits insulin signaling, interfering with its control of glucose homeostasis and metabolism. PTP1B activity is elevated in obesity and type 2 diabetes and is a major cause of insulin resistance. Trodusquemine (MSI-1436) is a "first-in-class" highly selective inhibitor of PTP1B that can cross the blood-brain barrier to suppress feeding and promote insulin sensitivity and glycemic control. Trodusquemine is a naturally occurring cholestane that can be purified from the liver of the dogfish shark, Squalus acanthias, but it can also be manufactured synthetically by a fairly laborious process that requires several weeks. Here, we tested a novel easily and rapidly (2 days) synthesized polyaminosteroid derivative (Claramine) containing a spermino group similar to Trodusquemine for its ability to inhibit PTP1B. Like Trodusquemine, Claramine displayed selective inhibition of PTP1B but not its closest related phosphatase TC-PTP. In cultured neuronal cells, Claramine and Trodusquemine both activated key components of insulin signaling, with increased phosphorylation of insulin receptor-ß (IRß), Akt and GSK3ß. Intraperitoneal administration of Claramine or Trodusquemine effectively restored glycemic control in diabetic mice as determined by glucose and insulin tolerance tests. A single intraperitoneal dose of Claramine, like an equivalent dose of Trodusquemine, suppressed feeding and caused weight loss without increasing energy expenditure. In summary, Claramine is an alternative more easily manufactured compound for the treatment of type II diabetes.

Targeting the disordered C terminus of PTP1B with an allosteric inhibitor.

PTP1B, a validated therapeutic target for diabetes and obesity, has a critical positive role in HER2 signaling in breast tumorigenesis. Efforts to develop therapeutic inhibitors of PTP1B have been frustrated by the chemical properties of the active site. We define a new mechanism of allosteric inhibition that targets the C-terminal, noncatalytic segment of PTP1B. We present what is to our knowledge the first ensemble structure of PTP1B containing this intrinsically disordered segment, within which we identified a binding site for the small-molecule inhibitor MSI-1436. We demonstrate binding to a second site close to the catalytic domain, with cooperative effects between the two sites locking PTP1B in an inactive state. MSI-1436 antagonized HER2 signaling, inhibited tumorigenesis in xenografts and abrogated metastasis in the NDL2 mouse model of breast cancer, validating inhibition of PTP1B as a therapeutic strategy in breast cancer. This new approach to inhibition of PTP1B emphasizes the potential of disordered segments of proteins as specific binding sites for therapeutic small molecules.

Molecular population dynamics of DNA structures in a bcl-2 promoter sequence is regulated by small molecules and the transcription factor hnRNP LL.

Minute difference in free energy change of unfolding among structures in an oligonucleotide sequence can lead to a complex population equilibrium, which is rather challenging for ensemble techniques to decipher. Herein, we introduce a new method, molecular population dynamics (MPD), to describe the intricate equilibrium among non-B deoxyribonucleic acid (DNA) structures. Using mechanical unfolding in laser tweezers, we identified six DNA species in a cytosine (C)-rich bcl-2 promoter sequence. Population patterns of these species with and without a small molecule (IMC-76 or IMC-48) or the transcription factor hnRNP LL are compared to reveal the MPD of different species. With a pattern recognition algorithm, we found that IMC-48 and hnRNP LL share 80% similarity in stabilizing i-motifs with 60 s incubation. In contrast, IMC-76 demonstrates an opposite behavior, preferring flexible DNA hairpins. With 120-180 s incubation, IMC-48 and hnRNP LL destabilize i-motifs, which has been previously proposed to activate bcl-2 transcriptions. These results provide strong support, from the population equilibrium perspective, that small molecules and hnRNP LL can modulate bcl-2 transcription through interaction with i-motifs. The excellent agreement with biochemical results firmly validates the MPD analyses, which, we expect, can be widely applicable to investigate complex equilibrium of biomacromolecules.

Synthesis and evaluation of some new aza-B-homocholestane derivatives as anticancer agents.

Using analogues of some marine steroidal oximes as precursors, a series of aza-B-homocholestane derivatives possessing different substituted groups at the 3-position of the steroidal nucleus were synthesized. Their biological activity against cancer cell proliferation was determined with multiple cancer cell lines. Aza-B-homocholestane derivatives possessing 3-hydroxyl, 3-hydroximino and 3-thiosemicarbazone groups displayed remarkable cytotoxicity to cancer cells via apoptosis inducing mechanism. Compounds 5, 10, 12, 15 and 18 exhibited better potency to inhibit cancer cell proliferation. In addition, compound 15 was further evaluated with three dimensional (3D) multicellular spheroids assay to determine its potency against spheroid growth. The structure-activity relationship (SAR) generated in the studies is valuable for the design of novel chemotherapeutic agents.

The transcriptional complex between the BCL2 i-motif and hnRNP LL is a molecular switch for control of gene expression that can be modulated by small molecules.

In a companion paper (DOI: 10.021/ja410934b) we demonstrate that the C-rich strand of the cis-regulatory element in the BCL2 promoter element is highly dynamic in nature and can form either an i-motif or a flexible hairpin. Under physiological conditions these two secondary DNA structures are found in an equilibrium mixture, which can be shifted by the addition of small molecules that trap out either the i-motif (IMC-48) or the flexible hairpin (IMC-76). In cellular experiments we demonstrate that the addition of these molecules has opposite effects on BCL2 gene expression and furthermore that these effects are antagonistic. In this contribution we have identified a transcriptional factor that recognizes and binds to the BCL2 i-motif to activate transcription. The molecular basis for the recognition of the i-motif by hnRNP LL is determined, and we demonstrate that the protein unfolds the i-motif structure to form a stable single-stranded complex. In subsequent experiments we show that IMC-48 and IMC-76 have opposite, antagonistic effects on the formation of the hnRNP LL-i-motif complex as well as on the transcription factor occupancy at the BCL2 promoter. For the first time we propose that the i-motif acts as a molecular switch that controls gene expression and that small molecules that target the dynamic equilibrium of the i-motif and the flexible hairpin can differentially modulate gene expression.

Sample preparation: a critical step in the analysis of cholesterol oxidation products.

In recent years, cholesterol oxidation products (COPs) have drawn scientific interest, particularly due to their implications on human health. A big number of these compounds have been demonstrated to be cytotoxic, mutagenic, and carcinogenic. The main source of COPs is through diet, and particularly from the consumption of cholesterol-rich foods. This raises questions about the safety of consumers, and it suggests the necessity for the development of a sensitive and a reliable analytical method in order to identify and quantify these components in food samples. Sample preparation is a necessary step in the analysis of COPs in order to eliminate interferences and increase sensitivity. Numerous publications have, over the years, reported the use of different methods for the extraction and purification of COPs. However, no method has, so far, been established as a routine method for the analysis of COPs in foods. Therefore, it was considered important to overview different sample preparation procedures and evaluate the different preparative parameters, such as time of saponification, the type of organic solvents for fat extraction, the stationary phase in solid phase extraction, etc., according to recovery, precision and simplicity.

Cytotoxic cholestane-type and ergostane-type steroids from the aerial parts of Euphorbia altotibetic.

Phytochemical investigation of the ethanol extract obtained from the aerial parts of the Euphorbia altotibetic PAULS. Grown in China resulted in the isolation of three new cholestane-type and three new ergostane-type steroids (cholest-5-en-2ß, 4ß-diol; cholest-5-en-1ß, 4ß-diol; cholest-5-en-1α, 3ß, 4α -triol; (22E)-ergosta-7,9,22-trien- 3ß-ol ß-D-glucoside; 5α-methoxy-(22E)-ergosta-7,9,22-trien-3ß-ol ß-D-glucoside; 6ß- methoxy-(22E)-ergosta-7,9,22-trien-3ß-ol ß-D-glucoside), along with seven known compounds. Their structures were established by extensive one- and two-dimensional NMR spectroscopy, as well as other spectrum and chemical analysis. The isolated new steroids exhibited potent anti-tumor activity against the HeLa cell and Hep-G2 cell with the 50% inhibiting concentration values ranging from 1.9 to 9.2 µg/mL.

New cholestane glycosides and sterols from the underground parts of Chamaelirium luteum and their cytotoxic activity.

Six new cholestane glycosides (1, 5, 6, 10, 12, and 13) and two new sterols (9 and 11), along with five known compounds (2-4, 7, and 8), were isolated from the underground parts of Chamaelirium luteum (Liliaceae). The structures of these new compounds were determined by spectroscopic analysis and the results of hydrolytic cleavage. The isolated compounds and aglycones were evaluated for their cytotoxic activity against HL-60 human leukemia cells. Compounds 6a, 10a, 12a, 13, and 13a were cytotoxic to HL-60 cells, with IC50 values of 12.8, 9.8, 15.3, 6.2, and 10.2 µM, respectively.