Exploration and biological evaluation of 20-vinyl pregnenes: A step forward toward selective modulators of the estrogen receptor α signaling for breast cancer treatment.

A series of D-ring modified steroids bearing a vinyl ketone pendant were synthesized and evaluated for antiproliferative activity against breast cancer cell line and cytochromes P450. The lead compound, 21-vinyl 20-keto-pregnene (2f) (IC50 = 2.4 µM), was shown to be a promising candidate for future anticancer drug design, particularly against estrogen receptor α (ERα)-positive breast cancer. The lead compound was found to have a significant effect on the signaling pathways in parental and 4-hydroxytamoxifen-resistant cells. Compound 2f modulated the ERK, cyclin D1, and CDK4 pathways and blocked the expression of ERα, the main driver of breast cancer growth. Compound 2f significantly reduced 17ß-estradiol-induced progesterone receptor expression. Accumulation of cleaved poly(ADP-ribose) polymerase in cells treated with compound 2f indicated induction of apoptosis. The selectivity analysis showed that lead compound 2f produces no significant effects on cytochromes P450, CYP19A1, CYP21A2, and CYP7B1.

Synthesis and biological evaluation of novel withangulatin A derivatives as potential anticancer agents.

Novel withangulatin A (WA) derivatives were synthesized and evaluated for antiproliferative activity against four human cancer cell lines (U2OS, MDA-MB-231, HepG2, and A549). Among these derivatives, 10 exhibited the most potent antiproliferative activity, with an IC50 value of 74.0 nM against the human breast cancer cell line MDA-MB-231 and potency that was 70-fold that of WA (IC50 = 5.22 µM). Moreover, 10 caused G2-phase cell cycle arrest in a concentration-dependent manner and induced the apoptosis of MDA-MB-231 cells by increasing intracellular reactive oxygen species (ROS). Compound 10 showed a high selectivity index (SI = 267.03) for breast cancer MDA-MB-231 cells. These results suggest that 10 is a promising anticancer agent.

A novel pregnene analogs: synthesis, cytotoxicity on prostate cancer of PC-3 and LNCPa-AI cells and in silico molecular docking study.

New pregnene analogs of N-hydroxamic acid 6, imino-propane hydrazides 7 and 8 as well as the aryl amides 9-11, oxadiazole, pyrazole and sulfinyl analogs 13-15, via the hydrazide analog 5 of methyl ((5-pregnen-3ß,17ß-diol-15α-yl)thio)propanoate (4) were synthesized. The in vitro cytotoxic activities of selected synthesized steroids against two human prostate cancer cell lines (PC-3, and LNCaP-AI) were evaluated by MTT assay. Compound 10 was the most active cytotoxic agent among these steroids against PC-3 and LNCaP-AI cell lines with inhibition of 96.2%, and 93.6% at concentration levels of 10.0 µM and 91.8%, and of 79.8% at concentration of 1.0 µM, respectively. Molecular docking study of 10 showed a hydrogen bonding with the amino acid Asn705 residue of the receptor 1E3G, together with hydrophobic interactions. Therefore, compound 10 can be considered as a promising anticancer agent due to its potent cytotoxic activity.

Discovery and optimization of withangulatin A derivatives as novel glutaminase 1 inhibitors for the treatment of triple-negative breast cancer.

To develop novel GLS1 inhibitors as effective therapeutic agents for triple-negative breast cancer (TNBC), 25 derivatives were synthesized from the natural inhibitor withangulatin A (IC50 = 18.2 µM). Bioassay optimization identified a novel and selective GLS1 inhibitor 7 (IC50 = 1.08 µM). In MDA-MB-231 cells, 7 diminished cellular glutamate levels by blocking glutaminolysis pathway, further triggering the generation of reactive oxygen species to induce caspase-dependent apoptosis. Molecular docking indicated that 7 interacted with a new reacting site of allosteric binding pocket by forming various interactions in GLS1. The intraperitoneal administration of 7 at a dose of 50 mg/kg exhibited remarkable therapeutic effects and no apparent toxicity in the MDA-MB-231 xenograft model, indicating its potential as a novel GLS1 inhibitor for treatment of TNBC.

Design and SAR of Withangulatin A Analogues that Act as Covalent TrxR Inhibitors through the Michael Addition Reaction Showing Potential in Cancer Treatment.

The thioredoxin system plays an important role in cancer cells. Inhibiting thioredoxin reductase (TrxR) has emerged as an effective strategy to selectively target cancer cells. Withangulatin A (WA), a natural product extracted from the whole herb of Physalis angulata L. (Solanaceae), exhibits potent anticancer activity and other diverse pharmacological activities. To improve activity and targeting, we designed and prepared 41 semisynthetic analogues of WA. Biological evaluation indicated that the most promising compound 13a displayed the most significant effect on HT-29 cells (human colon cancer cells) (IC50 = 0.08 µM). A structure-activity relationship study indicated that α,ß-unsaturated ketones and ester are necessary groups, allowing 13a to undergo Michael addition reactions with mercaptan and selenol. Liquid chromatography-mass spectrometry (LC-MS) analysis confirmed that 13a modified selenocysteine 498 (U) residues in the redox centers of TrxR, resulting in enzyme inhibition. Therefore, compound 13a acts as a novel TrxR inhibitor and may be a promising candidate for cancer intervention.

Antimalarial and cytotoxic activities of pregnene-type steroidal alkaloids from Holarrhena pubescens roots.

The phytochemical investigation of an alkaloidal extract of Holarrhena pubescens roots led to the isolation and identification of a new pregnene-type alkaloid, mokluangin D (1), together with nine known steroidal alkaloids (2-10). The structure of the new metabolite was determined on the basis of spectroscopic analyses including 1D- and 2D-NMR spectroscopy and mass spectrometry. Compounds 3 and 4 showed potent antimalarial activity against Plasmodium falciparum K1 stain with IC50 values of 1.2 and 2.0 µM, respectively, and showed weak cytotoxic activity against the NCI-H187 cell line with IC50 values of 27.7 and 30.6 µM, respectively. The substituent groups at C-3 and the carbonyl group at C-18 are important for the activity against the P. falciparum K1 stain.

Relative importance of phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK3/1) signaling during maturational steroid-induced meiotic G2-M1 transition in zebrafish oocytes.

Participation and relative importance of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling, either alone or in combination, have been investigated during 17α,20ß-dihydroxy-4-pregnen-3-one (DHP)-induced meiotic G2-M1 transition in denuded zebrafish oocyte. Results demonstrate that concomitant with rapid phosphorylation (activation) of Akt (Ser473) and MAPK (ERK1/2) at as early as 15 min of incubation, DHP stimulation promotes enhanced an GVBD response and histone H1 kinase activation between 1 and 5 h in full-grown oocytes in vitro. While p-Akt reaches its peak at 60 to 90 min and undergoes downregulation to the basal level by 240 min, ERK1/2 phosphorylation (activation) increases gradually until 120 min and remains high thereafter. Although, priming with MEK1/2 inhibitor U0126 is without effect, PI3K inhibitors, wortmannin or LY294002, delay the GVBD response significantly (P < 0.001) until 3 h but not at 5 h of incubation. Interestingly, blocking PI3K and MEK function together could abrogate steroid-induced oocyte maturation at all time points tested. While DHP stimulation promotes phospho-PKA catalytic (p-PKAc) dephosphorylation (inactivation) between 30-120 min of incubation, simultaneous inhibition of PI3K and MEK1/2 kinases abrogates DHP action. Conversely, elevated intra-oocyte cAMP, through priming with either adenylyl cyclase (AC) activator forskolin (FK) or dibutyryl cAMP (db-cAMP), abrogates steroid-induced Akt and ERK1/2 phosphorylation. Taken together, these results suggest that DHP-induced Akt and ERK activation precedes the onset of meiosis (GVBD response) in a cAMP-sensitive manner and PI3K/Akt and MEK/MAPK pathways together have a pivotal influence in the downregulation of PKA and resumption of meiotic maturation in zebrafish oocytes in vitro.

Comparative Proteomic Analysis of the Effect of Periplocoside P from Periploca sepium on Brush Border Membrane Vesicles in Midgut Epithelium of Mythimna separata Larvae.

Periplocoside P (PSP), a novel compound isolated from Periploca sepium Bunge, possesses insecticidal activity against some lepidopterans, such as Mythimna separata. In M. separata, the brush border membrane vesicles of the midgut epithelium are the initial site of action of periplocosides. We conducted two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry analysis to analyze differentially expressed proteins (DEPs) from periplocoside P (PSP)-treated M. separata. We successfully isolated seven up-regulated and three down-regulated DEPs that have been previously identified, as well as a novel DEP. The DEPs are implicated in protein degradation, transporter, folding, and synthesis, and in juvenile hormone biosynthesis. DEPs involved in the oxidative phosphorylation energy metabolism pathway are enriched. Through real-time polymerase chain reaction assay, we confirmed that vma1 expression is significantly up-regulated expression levels in PSP-treated M. separata larvae. Enzymology validation further indicated that PSP can significantly inhibit V-type ATPase activity in a concentration-dependent manner. Given these results, we speculate that in M. separata, the V-type ATPase A subunit in the midgut epithelium is the putative target binding site of periplocosides. This finding provides preliminary evidence for the mode of action of periplocosides.

Effects of Periplocoside P from Periploca sepium on the Midgut Transmembrane Potential of Mythimna separata Larvae.

Periplocoside P (PSP) isolated from the root bark of Periploca sepium contains a pregnane glycoside skeleton and possesses high insecticidal properties. Preliminary studies indicated that PSP disrupts epithelial functions in the midgut of lepidopteran larvae. In the present study, we examined the effects of PSP on the apical and basolateral membrane voltages, Va and Vbl, respectively, of cells from (1) midguts isolated from the larvae of the oriental armyworm Mythimna separata that were in vitro incubated with toxins and (2) midguts isolated from M. separata larvae force-fed with PSP. We compared the effects of PSP with the effects of the Bacillus thuringiensis toxin Cry1Ab and inactive periplocoside E (PSE) on the midgut epithelial cells. The results showed that Va rapidly decreased in the presence of PSP in a time- and dose-dependent manner, similar to the effects of Cry1Ab. By contrast, PSE did not affect the Va and Vbl. Additionally, PSP did not influence the Vbl. Given these results, we speculate that PSP may modulate transport mechanisms at the apical membrane of the midgut epithelial cells by inhibiting the V-type H+ ATPase.

Bioactive C21 Steroidal Glycosides from the Roots of Cynanchum otophyllum That Suppress the Seizure-like Locomotor Activity of Zebrafish Caused by Pentylenetetrazole.

Six new C21 steroidal glycosides, cynotophyllosides A-F (1-6), together with 16 known compounds, were isolated from the roots of Cynanchum otophyllum. The structures of the new compounds were elucidated by spectroscopic analysis and chemical methods. The three major components, otophylloside F (15), otophylloside B (17), and rostratamine 3-O-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-cymaropyranoside (18), suppressed the seizure-like locomotor activity caused by pentylenetetrazole in zebrafish. Preliminary structure-activity relation studies revealed that a pregnene skeleton with a C-12 ester group (ikemaoyl > cinnamoyl > hydroxy > p-hydroxybenzoyl) and a C-3 sugar chain consisting of three 2,6-dideoxysaccharide units is essential for this suppressive activity.

Complete NMR assignments of undegraded asterosaponins from Asterias amurensis.

Four asterosaponins, thornasteroside A (1), versicoside A (2), anasteroside B (3), and asteronylpentaglycoside sulfate (4), were isolated from the predatory starfish Asterias amurensis Lütken. Unlike previous studies focusing on structure elucidation by degradation of the complex saponin molecules, complete nuclear magnetic resonance (NMR) assignment for the intact molecules was accomplished using 600 MHz high magnetic field NMR. The complete set of NMR assignments can help in the structure elucidation of asterosaponins isolated in low yields without resorting to chemical degradation. Furthermore, this approach can be extended to other complex steroidal saponins, which may accelerate the discovery of bioactive secondary metabolites from this invasive starfish species.

Synthesis and antimetastatic effects of E-salignone amide derivatives.

The preparation of novel E-salignone derivatives and their biological evaluation as potential antimetastatic agents is described. The E-salignone amide derivatives were prepared from epiandrosterone and androsterone, and characterized by analytical (1) H NMR, (13) C NMR, and mass spectrometry. The derivatives were evaluated for antimetastatic activity in MDA-MB-231 cells by using a transwell assay. Comparing with the positive control, LY294002, compounds 19b, 19d, and 19e exhibited significant inhibitory effects on the EGF-induced invasion of MB-MDA-231 cells. Moreover, compound 19b also had antimigration effects in wound-healing assay. Compound 19b may represent a novel antimetastatic agent for treating breast cancer.

The guggulsterone derivative GG-52 inhibits NF-κB signaling in bone marrow-derived dendritic cells and attenuates colitis in IL-10 knockout mice.

AIMS: We previously demonstrated that the novel guggulsterone derivative guggulsterone-52 (GG-52) inhibited the activation of nuclear factor (NF)-κB signaling in intestinal epithelial cells and had preventive and therapeutic effects on dextran sulfate sodium-induced acute colitis. This study investigates the anti-inflammatory effects of GG-52 on bone marrow-derived dendritic cells (BMDCs) and chronic colitis in IL-10(-/-) mice. MAIN METHODS: BMDCs were generated from the femurs of C57BL/6 wild-type and IL-10(-/-) mice. BMDCs were stimulated with lipopolysaccharide (LPS) in the presence or absence of GG-52. The effect of GG-52 on NF-κB signaling in BMDCs was examined by real-time RT-PCR for IL-12p40 and TNF-α gene expression, western blotting for IκBα degradation, and electrophoretic mobility shift assay. For in vivo studies, wild-type or IL-10(-/-) mice were treated with or without GG-52. Colitis was quantified by the evaluation of histopathological findings. Double immunofluorescence staining for CD11c and phosphorylated IκB kinase (IKK)-α was performed to detect IKK activation in DCs in colonic tissue. KEY FINDINGS: GG-52 significantly inhibited LPS-induced IL-12p40 and TNF-α gene expression, IκBα degradation, and NF-κB DNA binding activity in BMDCs. In the IL-10(-/-) mouse model chronic colitis, administration of GG-52 significantly reduced the severity of colitis as assessed by histopathology, and suppressed IKK activation in DCs in colonic tissue. SIGNIFICANCE: These results indicate that the novel guggulsterone derivative GG-52 blocks NF-κB activation in BMDCs and ameliorates chronic colitis in IL-10(-/-) mice, which suggest that GG-52 is a potential therapeutic agent for inflammatory bowel diseases.

03219A, a new Δ(8,9)-pregnene isolated from Streptomyces sp. SCSIO 03219 obtained from a South China Sea sediment.

03219A (1), a new pregnene steroid possessing a rare Δ(8,9)-double bond in the skeleton, together with the known naphthoquinone antibiotic (+)-cryptosporin (2) have been isolated from the fermentation broth of Streptomyces sp. SCSIO 03219, which was isolated from a marine sediment collected in the South China Sea. The structure of 03219A was elucidated using a combination of NMR, MS and X-ray crystallographic methods.

The guggulsterone derivative GG-52 inhibits NF-κB signaling in gastric epithelial cells and ameliorates ethanol-induced gastric mucosal lesions in mice.

Gastric mucosal inflammation can develop after challenge with noxious stimuli such as alcohol. Specially, alcohol stimulates the release of inflammatory cytokines but does not increase gastric acid secretion, leading to gastric mucosal damage. The plant sterol guggulsterone and its novel derivative GG-52 have been reported to inhibit nuclear factor-κB (NF-κB) signaling in intestinal epithelial cells and experimental colitis. In the present study, we investigated the anti-inflammatory effects of GG-52 on gastric epithelial cells and on ethanol-induced gastric mucosal inflammation in mice. GG-52 inhibited the expression of interleukin-8 (IL-8) in gastric epithelial AGS and MKN-45 cell lines stimulated with tumor necrosis factor (TNF)-α in a dose-dependent manner. Pretreatment with GG-52 suppressed TNF-α-induced activation of IκB kinase (IKK) and NF-κB signaling in MKN-45 cells. In contrast, the inactive analog GG-46 did not produce significant changes in IL-8 expression or NF-κB activation. In a model of ethanol-induced murine gastritis, administration of GG-52 significantly reduced the severity of gastritis, as assessed by macroscopic and histological evaluation of gastric mucosal damage. In addition, the ethanol-induced upregulation of chemokine KC, a mouse homolog of IL-8, and phosphorylated p65 NF-κB signals were significantly inhibited in murine gastric mucosa pretreated with GG-52. These results indicate that GG-52 suppresses NF-κB activation in gastric epithelial cells and ameliorates ethanol-induced gastric mucosal lesions in mice, suggesting that GG-52 may be a potential gastroprotective agent.

Chemical constituents from the roots of Periploca sepium with insecticidal activity.

Five compounds were isolated from the root powder of Periploca sepium. By mainly HR-ESI-MS, (1)H, (13)C, and 2D NMR spectral data, they were characterized as periplocoside X (1), oligasaccharide A (2), periplocoside A (3), periplocoside E (4), and periplocoside N (5), respectively. Compounds 1-5 were found to possess insecticidal activities against the red imported fire ant. Among the compounds, periplocoside X showed significant activity with LD(50) values of 748.99, 116.62, 2169.58, and 3079.33mg/l against soldiers, workers, males, and alate females of red imported fire ant, respectively.

A new pregnane glycoside and oligosaccharide from Parabarium huaitingii.

Two new compounds, along with two known compounds, were isolated from the barks of Parabarium huaitingii, and their structures were determined as 5α-pregn-6-ene-3ß,17α,20(S)-triol-20-O-ß-d-digitoxopyranoside (1), cymaropyranurolactone 4-O-ß-d-digitalopyranosyl-(1 â†’ 4)-O-ß-d-cymaropyranosyl-(1 â†’ 4)-O-ß-d-oleandropyranosyl-(1 â†’ 4)-O-ß-d-cymaropyranoside (2), 3ß,17α,20(S)-trihydroxy-5α-pregn-6-ene (3), and 5α-pregn-6-ene-3ß,17α,20(S)-triol-3-O-ß-d-digitalopyranoside (4) by spectroscopic methods.

Neurosteroid analogues. 16. A new explanation for the lack of anesthetic effects of δ(16)-alphaxalone and identification of a δ(17(20)) analogue with potent anesthetic activity.

This study addresses the hypothesis that the lack of anesthetic activity for (3α,5α)-3-hydroxypregn-16-ene-11,20-dione (Δ(16)-alphaxalone) is explained by the steroid Δ(16) double bond constraining the steroid 20-carbonyl group to a position that prevents it from favorably interacting with γ-aminobutyric acid type A (GABA(A)) receptors. A series of Δ(16) and Δ(17(20)) analogues of Δ(16)-alphaxalone was prepared to evaluate this hypothesis in binding, electrophysiological, and tadpole anesthesia experiments. The results obtained failed to support the hypothesis. Instead, the results indicate that it is the presence of the C-21 methyl group in Δ(16)-alphaxalone, not the location of the constrained C-20 carbonyl group, that prevents Δ(16)-alphaxalone from interacting strongly with the GABA(A) receptor and having anesthetic activity. Consistent with this conclusion, a Δ(17(20)) analogue of Δ(16)-alphaxalone without a C-21 methyl group was found to be very similar to the anesthetic steroid (3α,5α)-3-hydroxypregnane-11,20-dione (alphaxalone) with regard to time of onset and rate of recovery from anesthesia when administered to mice by tail vein injection.

Withagulatin A inhibits hepatic stellate cell viability and procollagen I production through Akt and Smad signaling pathways.

AIM: To investigate the effects of the natural product Withagulatin A on hepatic stellate cell (HSC) viability and type I procollagen production. The potential mechanism underlying the pharmacological actions was also explored. METHODS: The effect of Withagulatin A on cell viability was evaluated in HSC and LX-2 cells using a sulforhodamine B (SRB) assay. Cell cycle distribution was analyzed using flow cytometry. Type I procollagen gene expression was determined using real-time PCR. Regulation of signaling molecules by Withagulatin A was detected using Western blotting. RESULTS: Primary rat HSCs and the human hepatic stellate cell line LX-2 treated with Withagulatin A (0.625-20 micromol/L) underwent a dose-dependent decrease in cell viability, which was associated with S phase arrest and the induction of cell apoptosis. In addition, the natural product decreased phosphorylation of the Akt/mTOR/p70S6K pathway that controls cell proliferation and survival. Furthermore, Withagulatin A (1, 2 mumol/L) inhibited transforming growth factor-beta (TGF-beta) stimulated type I procollagen gene expression, which was attributable to the suppression of TGF-beta stimulated Smad2 and Smad3 phosphorylation. CONCLUSION: Our results demonstrated that Withagulatin A potently inhibited HSC viability and type I procollagen production, thereby implying that this natural product has potential use in the development of anti-fibrogenic reagents for the treatment of hepatic fibrosis.

Homo- and heptanor-sterols and tremulane sesquiterpenes from cultures of Phellinus igniarius.

Four steroids, a homopregnene (1) and three heptanorergosterane derivatives (2-4), nine tremulane sesquiterpenes (5-13), and 18 known compounds have been isolated from cultures of the fungus Phellinus igniarius. Their structures and absolute configurations were elucidated by spectroscopic data analysis. In preliminary in vitro assays, at 10(-5) M, compounds 8, 9, 13, and 3beta-hydroxy-11,12-O-isopropyldrimene (14) showed significant vascular-relaxing activities against phenylephrine-induced vasoconstriction with relaxing rates of 35.7%, 45.4%, 46.6%, and 32.1%, respectively, as compared with the blank control.