Isolation of a spirolactone norditerpenoid as a yeast Ca2+ signal transduction inhibitor from Kuji amber and evaluation of its effects on PPM1A activity.

A different type of biologically active compound from Kuji amber (Late Cretaceous, Japan) before the K-Pg boundary [65 million years ago (Ma)] was isolated based on the growth-restoring activity of a mutant yeast involving Ca2+ signal transduction. It was identified as a spirolactone norditerpenoid, (4R*, 5S*, 8R*, 9R*, 10S*)-14,15,16,19-tetranor-labdan-13,9-olide (1) from spectral analyses with high-resolution electron ionization mass spectrometry (HREIMS), 1D and 2D nuclear magnetic resonance (NMR). Although the planar structure of 1 is known as an artificial derivative from marrubiin, it was isolated as a natural product from Kuji amber and its structure was elucidated for the first time. It had a growth-restoring activity against the mutant yeast through the direct or indirect inhibition of calcineurin activity [protein phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A) activation]. Furthermore, the compound had potent inhibitory effect against the degranulation of rat basophilic leukemia 2H3 (RBL-2H3) cells.

Epimeric spirolactone-type triterpenoids from Abies faxoniana Rehd.

Phytochemical investigation of Abies faxoniana Rehd. led to the isolation of two pairs of new epimeric spirolactone-type triterpenoids (1/1' and 2/2') and 11 known terpenoids (3-13). Compounds 1/1' and 2/2' were isolated as epimeric mixtures due to the C-23 ketal tautomerism in their spirolactone structures. The dynamic HPLC manifested that the C-23 epimeric mixtures interconverted into each other in solution. Structure determinations were based on extensive NMR and HRESIMS spectroscopic analysis. Meanwhile, their cytotoxic activities were tested by MTT method. Compound 5 showed cytotoxicities against MCF-7 and A549 cells with IC50 values of 6.5 and 5.7µM, respectively. Compounds 1/1' had IC50 values of 10.0 and 12.3µM for Huh7 and SMMC7721 cells, respectively.

Simultaneous separation and rapid determination of spironolactone and its metabolite canrenone in different pharmaceutical formulations and urinary matrices by capillary zone electrophoresis.

The aim of this study was to develop a novel, sensitive, precise, simple, and rapid capillary zone electrophoresis method for the quality control of spironolactone in three different formulation types and a rapid simultaneous determination of the content of spironolactone and canrenone in urine samples using fluocinonide as an internal standard. After optimization of separation conditions, the electrolyte solution was the pH 5.5, 20 mM phosphate buffer containing 4.5 g/L sulfated-ß-cyclodextrin, 15 kV of electric filed across the capillary applied at 25°C. A diode array detector was used, and the detection wavelength was 260 nm. Under optimum conditions, good linearity was achieved with correlation coefficients from 0.9976 to 0.9997. Detection limits were 0.56 and 0.20 µg/mL, and the quantitation limits were 1.87 and 0.67 µg/mL, respectively. Excellent accuracy and precision were obtained. Recoveries of the analytes varied from 100.8 to 103.1%. The results indicated that baseline separation of analytes was obtained and this method was suitable for quantitative determination of spironolactone in pharmaceutical preparations and rapid simultaneous determination of the content of spironolactone and its major metabolite canrenone in urine samples.

Stability and removal of spironolactone from wastewater.

Stability and removal of spironolactone (SP) from wastewater produced at Al-Quds University Campus were investigated. Kinetic studies on both pure water and wastewater coming from secondary treatment (activated sludge) demonstrated that the potassium-sparing diuretic (water pill), spironolactone, underwent degradation to its hydrolytic derivative, canrenone, in both media. The first-order hydrolysis rate of SP in activated sludge at 25°C (3.80 × 10(-5) s(-1)) was about 49-fold larger than in pure water (7.4 × 10(-7) s(-1)). The overall performance of the wastewater treatment plant (WWTP) installed in the University Campus was assessed showing that more than 90% of spiked SP was removed together with its newly identified metabolites. In order to look for a technology to supplement or replace ultra-filtration membranes, the effectiveness of adsorption and filtration by micelle-clay filters for removing SP was tested in comparison with activated charcoal. Batch adsorption in aqueous suspensions was well described by Langmuir isotherms, showing a better removal by the micelle-clay material. Filtration of SP water solutions by columns filled with a mixture of sand and a micelle-clay complex showed complete removal of the drug at concentrations higher than in sand/activated-charcoal filled filters.

Yaoshanenolides A and B: new spirolactones from the bark of Machilus yaoshansis.

Two novel tricyclic spirolactones bearing long linear alkyl chains, yaoshanenolides A (1) and B (2), formed by Diels-Alder[4 + 2] cycloaddition of a molecule of each butenolide with ß-phellandrene, were isolated from the bark of Machilus yaoshansis. Their structures and absolute configurations were determined by extensive spectroscopic methods, especially 2D NMR and ECD data analysis. The proposed biosynthetic pathway is discussed. Both compounds exhibited nonselective cytotoxic activities against several human cancer cell lines.

Structural basis of spirolactone recognition by the mineralocorticoid receptor.

Spirolactones are potent antagonists of the mineralocorticoid receptor (MR), a ligand-induced transcription factor belonging to the nuclear receptor superfamily. Spirolactones are synthetic molecules characterized by the presence of a C17 gamma-lactone, which is responsible for their antagonist character. They harbor various substituents at several positions of the steroid skeleton that modulate their potency in ways that remain to be determined. This is particularly obvious for C7 substituents. The instability of antagonist-MR complexes makes them difficult to crystallize. We took advantage of the S810L activating mutation in MR (MR(S810L)), which increases the stability of ligand-MR complexes to crystallize the ligand-binding domain (LBD) of MR(S810L) associated with 7alpha-acetylthio-17beta-hydroxy-3-oxopregn-4-en-21-carboxylic acid gamma-lactone (SC9420), a spirolactone with a C7 thioacetyl group. The crystal structure makes it possible to identify the contacts between SC9420 and MR and to elucidate the role of Met852 in the mode of accommodation of the C7 substituent of SC9420. The transactivation activities of MR(S810L/Q776A), MR(S810L/R817A), and MR(S810L/N770A) reveal that the contacts between SC9420 and the Gln776 and Arg817 residues are crucial to maintaining MR(S810L) in its active state, whereas the contact between SC9420 and the Asn770 residue contributes only to the high affinity of SC9420 for MR. Moreover, docking experiments with other C7-substituted spirolactones revealed that the MR(S810L)-activating potency of spirolactones is linked to the ability of their C7 substituent to be accommodated in LBD. It is remarkable that the MR(S810L)-activating and MR(WT)-inactivating potencies of the C7-substituted spirolactones follow the same order, suggesting that the C7 substituent is accommodated in the same way in MR(S810L) and MR(WT). Thus, the MR(S810L) structure may provide a powerful tool for designing new, more effective, MR antagonists.

Development and validation of an HPLC method for the determination of spironolactone and its metabolites in paediatric plasma samples.

An HPLC method has been developed and validated for the determination of spironolactone, 7 alpha-thiomethylspirolactone and canrenone in paediatric plasma samples. The method utilises 200 microl of plasma and sample preparation involves protein precipitation followed by Solid Phase Extraction (SPE). Determination of standard curves of peak height ratio (PHR) against concentration was performed by weighted least squares linear regression using a weighting factor of 1/concentration2. The developed method was found to be linear over concentration ranges of 30-1000 ng/ml for spironolactone and 25-1000 ng/ml for 7 alpha-thiomethylspirolactone and canrenone. The lower limit of quantification for spironolactone, 7 alpha-thiomethylspirolactone and canrenone were calculated as 28, 20 and 25 ng/ml, respectively. The method was shown to be applicable to the determination of spironolactone, 7 alpha-thiomethylspirolactone and canrenone in paediatric plasma samples and also plasma from healthy human volunteers.

Isolation and identification of novel impurities in spironolactone.

Three known and five new steroidal compounds as impurities in spironolactone were isolated from the enriched mother liquor by using various chromatographic methods. Their structures were elucidated by spectrometric analysis. New compounds were characterized as 3-(3,3-dimethoxy-5 alpha,7 alpha-epidithio-17beta-hydroxy-4-androstan-17 alpha-yl) propionic acid gamma-lactone (6); 3-(3-oxo-7 alpha-acetylthio-6 beta,17beta-dihydroxy-4-androsten-17 alpha-yl) propionic acid gamma-lactone (7); 7 alpha-acetylthio-17beta-20-isopropylidendioxy-21-nor-17 alpha-pregn-4-en-3-one (8); 7 alpha-acetylthio-3-oxo-pregna-4,17(20)i-dien-22-oic acid methyl ester (9) and 7 alpha-acetylthio-17-methyl-18-nor-androsta-4-en-3-one (10).

Novel spironolactone-analogs as impurities in spironolactone.

Six novel spironolactone-analogs steroids (3-8) were isolated from spironolactone by using various chromatographic methods. Their structures were elucidated by spectrometric analysis. Two of the analogs (3 and 7) were confirmed by X-ray crystallography. The A-ring of compounds 3-7 is opened at C-2C-3 bond, and compound 7 is an organic polysulfide, which has a rare, nine-membered ring with a five sulfur atom bridge.

The discovery, isolation and identification of aldosterone: reflections on emerging regulation and function.

This paper has a focus on the early history of aldosterone. The Taits take us on a chronological trawl through the history in which they had a first hand role and made a major contribution-their bioassay was in many ways the key. The gifted Swiss chemists made a critical contribution to the scale and isolation of larger amounts. This was international collaboration at its best. Developing technologies were utilised as crucial cutting edge applications in the advancing front, technology transfer before the word was invented. Measurement of aldosterone and angiotensin were crucial advances to the understanding of the regulation of the hormone. In the period 1960-2003, some 30,000 papers mentioned aldosterone as a keyword, even so advances on a larger scale were slow. I have indicated some of my own work with the Howard Florey team using the adrenal autotransplant in the conscious sheep. Recently, the understanding of the role of induced proteins, the flow on from the RALES trial and the development of eplerenone has revitalised the aldosterone field.

A bioactive spirolactone iridoid and triterpenoids from Himatanthus sucuuba.

Himatanthus sucuuba is an Amazonian tree with abundant, yet conflicting ethnobotanical information. Investigation of the polar and non-polar constituents led to the isolation of plumericin, a bioactive spirolactone iridoid, and four known pentacylic triterpenes: lupeol acetate, lupeol cinnamate, lupeol beta-phenyl propionate, and alpha-amyrin cinnamate.

Isolation and identification of a sulfur-containing metabolite of spironolactone from human urine.

In the urine of normal subjects who were given an oral dose of 500 mg spironolactone (3-(3-oxo-7alpha-acetylthio-17beta-hydroxy-4-androsten-17alpha-yl)-propionic acid gamma-lactone; Aldactone) together with 100uCi H-20, 21 spironolactone, a so far unknown major metabolite has been detected by thin layer chromatography. The metabolite then could be isolated by means of counter-current-distribution. According to masspectral and magnetic resonance data, the metabolite has been assigned the structure of 3-(3-oxo-7alpha-methyl sulfonyl-6beta, 17beta-dihydroxy-4-androsten-17alpha-yl)-propionic acid gamma-lactone. By oxidation of the corresponding methylsulfinyl compound - another already known metabolite of spironolactone - with m-chloroperbenzoic acid, a compound has been isolated which proved to be identical with the new metabolite according to TLC, MS and NMR.