Padma Marwah

Ergosteroids II: Biologically Active Metabolites and Synthetic Derivatives of Dehydroepiandrosterone

An improved procedure for the synthesis of 3 beta-hydroxyandrost-5-ene-7,17-dione, a natural metabolite of dehydroepiandrosterone (DHEA) is described. The synthesis and magnetic resonance spectra of several other related steroids are presented. Feeding dehydroepiandrosterone to rats induces enhanced formation of several liver enzymes among which are mitochondrial sn-glycerol 3-phosphate dehydrogenase (GPDH) and cytosolic malic enzyme. The induction of these two enzymes, that complete a thermogenic system in rat liver, was used as an assay to search for derivatives of DHEA that might be more active than the parent steroid. Activity is retained in steroids that are reduced to the corresponding 17 beta-hydroxy derivative, or hydroxylated at 7 alpha or 7 beta, and is considerably enhanced when the 17-hydroxy or 17-carbonyl steroid is converted to the 7-oxo derivative. Several derivatives of DHEA did not induce the thermogenic enzymes whereas the corresponding 7-oxo compounds did. Both short and long chain acyl esters of DHEA and of 7-oxo-DHEA are active inducers of the liver enzymes when fed to rats. 7-Oxo-DHEA-3-sulfate is as active as 7-oxo-DHEA or its 3-acetyl ester, whereas DHEA-3-sulfate is much less active than DHEA. Among many steroids tested, those possessing a carbonyl group at position 3, a methyl group at 7, a hydroxyl group at positions 1, 2, 4, 11, or 19, or a saturated B ring, with or without a 4-5 double bond, were inactive.

Ergosteroids. VI. Metabolism of dehydroepiandrosterone by rat liver in vitro: a liquid chromatographic-mass spectrometric study.

Because relatively large amounts of dehydroepiandrosterone (DHEA) are required to demonstrate its diverse metabolic effects, it is postulated that this steroid may be converted to more active molecules. To search for the possible receptor-recognized hormones. DHEA was incubated with whole rat liver homogenate and metabolite appearances were studied by LC-MS as a function of time to predict the sequence of their formation. An array of metabolites has been resolved, identified and characterized by highly specific and accurate technique of LC-MS, and several of these steroids were analyzed quantitatively. Their identities were established by comparison with pure chemically synthesized compounds and by chemical degradation of isolated fractions. In the present study, we have reasonably established that DHEA was converted to 7alpha-OH-DHEA, 7-oxo-DHEA, and 7beta-OH-DHEA in sequence. These metabolites were further reduced at position 7 and/or 17 to form their respective diols and triols, which were also sulfated at 3beta-position. DHEA and its 7-oxygenated derivatives were also converted to their respective 3beta-sulfate esters. Several of these steroids are being reported for the first time. 16Alpha-hydroxy-DHEA, androst-5-ene-3beta,16alpha,17beta-triol, androst-4-ene-3,17-dione, 11-hydroxy-androst-4-ene-3,17-dione, androst-5-ene-3,17-diol and testosterone were also identified and characterized. In all, 19 metabolites of DHEA are being reported in this extensive study. We have also detected the formation of 12 additional metabolites including several conjugates, which are the subject of current investigation.

An economical and green approach for the oxidation of olefins to enones

A new and optimized water-based procedure for the oxidation of steroidal olefins and benzylic compounds to α,β-enones, using household laundry bleach and aqueous tert-butyl hydroperoxide at sub-ambient temperature, is described. This simple methodology is not only economical but is also environmentally friendly. Issues of reactivity, selectivity and scope of the reaction were investigated with a variety of steroidal olefins and benzylic compounds.

3β-Acetoxyandrost-1,5-diene-17-ethylene ketal functions as a potent antiandrogen with marginal agonist activity

The majority of available antiandrogens have been reported to possess agonist activity to induce prostate-specific antigen, which might result in antiandrogen withdrawal syndrome. Here we report the identification of 3β-acetoxyandrost-1,5-diene-17-ethylene ketal (ADEK) from dehydroepiandrosterone metabolites and derivatives as a potent antiandrogen. We found ADEK could interrupt androgen binding to the androgen receptor (AR) and suppress androgen-induced transactivations of WT AR and a mutant AR in prostate cancer cells. ADEK inhibited prostate-specific antigen expression as well as growth in LNCaP prostate cancer cells stimulated by androgen. Importantly, ADEK had only marginal agonist effects, as compared with commonly used antiandrogens such as hydroxyflutamide and bicalutamide, leading to a lower possibility of inducing withdrawal response. Moreover, ADEK could block an adrenal androgen androstenediol-induced AR transactivation that hydroxyflutamide and bicalutamide failed to block. These unique antiandrogenic activities make ADEK a potential therapeutic compound that might be able to inhibit AR-mediated prostate cancer progression. Further in vivo studies might facilitate the development of a better antiandrogen for the treatment of prostate cancer.

Liquid chromatography-electrospray ionization mass spectrometric analysis of corticosterone in rat plasma using selected ion monitoring.

A simple and fast yet highly sensitive and specific method based on HPLC coupled to electrospray ionization mass spectrometry has been developed for the quantitation of corticosterone in rat plasma. After extraction of rat plasma (100 microl) with diethyl ether using 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (Sigma) as internal standard, HPLC was performed on a short C8 column (Zorbax-Eclipse, 50x4.6 mm I.D.) using a steep methanol-water gradient (methanol 54% to 90% in 6 min). Detection was performed on a single quadruple mass spectrometer in selected ion monitoring mode (m/z 369 for corticosterone and 364 for the internal standard). The detection limit of the assay was 9 fmol (3 pg) of corticosterone on column. In vitro data were subjected to curve fitting (cubic, r2=0.9999). Recovery of corticosterone after extraction ranged from 81 to 93%. The relative standard deviations for intra- and inter-assay precision ranged from 0.8 to 3.6% and 5.2 to 12.9%, respectively. Corticosterone did not undergo any appreciable degradation when stored in plasma at -20 degrees C for 2 months. The assay is routinely used in our laboratory to examine corticosterone levels as a marker of stress in rats and may also be used for the determination of 18-hydroxy-11-deoxycorticosterone.

Microwave induced selective enolization of steroidal ketones and efficient acetylation of sterols in semisolid state

Under microwave irradiation steroidal enones, more specifically, position three carbonyls were efficiently and selectively converted to the corresponding enol acetates in the presence of additional enolizable carbonyl functions at other positions, using acetic anhydride and a catalytic amount of toluene-p-sulfonic acid. Acetylation of hydroxyl groups of the sterols, including those at the hindered positions, was near quantitative. Strictly anhydrous conditions were not a pre-requisite for acetylation and the reaction system easily tolerated up to 10% (v/v) moisture.

High-performance liquid chromatographic analysis of dehydroepiandrosterone

Qualitative and quantitative analysis of dehydroepiandrosterone and its conjugates in biological matrices and establishment of their relationships with physiological functions is a very active field. This review article discusses methods of separation and quantification of dehydroepiandrosterone and its conjugates using high-performance liquid chromatographic techniques.

C19-5-ene steroids in nature

Dehydroepiandrosterone (DHEA), produced from cholesterol in the adrenals, is the most abundant steroid in our circulation. It is present almost entirely as the sulfate ester, but the free steroid is the form that serves as a precursor of estrogens and androgens, as well as 7- and 16-oxygenated derivatives. Mammalian tissues reduce the 17-keto Group of DHEA to produce androstenediol-a weak estrogen and full-fledged androgen. Its androgen activity is not inhibited by the anti-androgens commonly used to treat prostate cancer. It is probably responsible for the growth of therapy-resistant prostate cancer. DHEA is hydroxylated at the 7 alpha position, and this derivative is oxidized by 11 beta-hydroxysteroid dehydrogenase to form 7-keto DHEA. The latter is reduced by the same dehydrogenase to form 7 beta-hydroxy DHEA. When fed to rats, each of the latter three steroids induce the formation of two thermogenic enzymes in the liver. The late-term human fetus produces relatively large amounts of 16 alphahydroxy DHEA, which serves the mother as a precursor of estriol.

Transformations of DHEA and its metabolites by rat liver

Because dehydroepiandrosterone (DHEA) has a wide variety of weak beneficial effects in experimental animals and humans, we searched for metabolites of this steroid in the hope of finding more active compounds that might qualify for the title “steroid hormone”. Incubation of DHEA with rat liver homogenate fortified with energy-yielding substrates resulted in rapid hydroxylation at the 7α-position of the molecule and subsequent conversion to other 7-oxygenated steroids in the sequence DHEA»7α-hydroxyDHEA»7-oxoDHEA»7β-hydroxyDHFA, with branching to diols, triols, and sulfate esters. The ability of these metabolites to induce the formation of liver thermogenic enzyme activity increased from left to right in that sequence. A total of 25 different steroids were characterized, and at least six additional structures that are currently under study were produced from DHEA. 7-OxoDHEA is more effective than DHEA in enhancing memory performance in old mice and in reversing the amnesic effects of scopolamine.

Ergosteroids IV: synthesis and biological activity of steroid glucuronosides, ethers, and alkylcarbonates

The 7-oxo derivative of dehydroepiandrosterone is more active than the parent steroid and is devoid of adverse side effects in rats, monkeys and humans. In anticipation of possible therapeutic use we have sought more active, longer lasting forms of 7-oxo- and 7beta-hydroxydehydroepiandrosterones. The 7-oxo- and 7-hydroxy steroids have been converted to glucuronides, ethers and carbonate esters. The syntheses of these compounds are described and their ability to induce the formation of liver thermogenic enzymes when fed to rats is reported. Some of the new derivatives were found to be somewhat more effective than the equimolar amounts of 7-oxo-DHEA with which they were compared in each experiment.