Antibacterial Δ(1) -3-ketosteroids from the South China Sea gorgonian coral Subergorgia rubra.

Three new Δ(1) -3-ketosteroids characterized with a 9-OH, subergosterones A-C (1-3), together with five known analogs 4-8, were obtained from the gorgonian coral Subergorgia rubra collected from the South China Sea. The structures of 1-3, including their absolute configurations, were determined by comprehensive spectroscopic methods and electronic circular dichroism (ECD) experiments. Compounds 2 and 3 exhibited inhibitory antibacterial activities against Bacillus cereus with MIC values of 1.56 µM.

Design, synthesis and biological evaluation of novel steroidal spiro-oxindoles as potent antiproliferative agents.

Two series of novel steroidal spiro-pyrrolidinyl oxindoles 3a-t and 6a-c were designed and synthesized from dehydroepiandrosterone using the 1,3-dipolar cycloaddition as the key step and further evaluated for their antiproliferative activities for four human cancer cell lines (MGC-803, EC109, SMMC-7721 and MCF-7). This protocol achieved the formation of two CC bonds, one CN bond and the creation of one new five-membered pyrrolidine ring and three contiguous stereocenters in a single operation. Biological evaluation showed that these synthesized steroidal spiro-pyrrolidinyl oxindoles possessed moderate to good antiproliferative activities against the tested cell lines and some of them were more potent than 5-Fu. Particularly, compound 3g showed good antiproliferative activity against SMMC-7721 (IC50=0.71µM). Steroid dimer 6b showed improved antiproliferative activities against SMMC-7721 and MCF-7 with the IC50 values of 4.30 and 2.06µM, respectively. Flow cytometry analysis demonstrated that compound 3n caused the cellular early apoptosis and cell cycle arrest at G2/M phase in a concentration- and time-dependent manner. [Corrected]

Steroids with inhibitory activity against the prostate cancer cells and chemical diversity of marine alga Tydemania expeditionis.

A new diketosteroid, (E)-stigmasta-24(28)-en-3,6-dione (1), along with three known steroids (2-4) was isolated from marine alga Tydemania expeditionis collected in China Sea. Their structures were elucidated by extensive spectroscopic methods. Comparison of the chemical constituents revealed significant diversity among different locations. The biological activities of 1, 3 and 4 were evaluated on the prostate cancer cell lines and androgen receptor. Compound 1 exhibited moderate inhibitory activities against the prostate cancer cells DU145, PC3 and LNCaP with IC(50) values of 31.27±1.50, 40.59±3.10 and 19.80±3.84 µM, respectively. Compound 3 showed more potent activities with IC(50) values of 12.38±2.47, 2.14±0.33 and 1.38±0.07 µM, respectively. However, compound 4 showed only weak inhibitory activities on LNCaP cells and was inactive on DU145 and PC3 cells. A competitive binding assay showed that compound 1 exhibited significant affinity to the androgen receptor with an IC(50) value of 7.19±0.45 µM, while 3 and 4 were inactive. The fact that the inhibitory properties of 1 and 3 against the prostate cancer cells were inconsistent with their affinities to the androgen receptor suggested that there might be other mechanism of action involved in the cytotoxic activity.

Antigenotoxic ketosteroid from the red algae Jania adhaerens.

A new ketosteroid, 6ß,16ß-dihydroxycholest-4-en-3-one (1), in addition to the known 6ß-hydroxycholest-4-en-3-one (2), 6ß-hydroxycholest-4,22-dien-3-one (3) and 16ß-hydroxy-5α-cholestan-3,6-dione (4), was isolated from the red alga Jania adhaerens. The structures were assigned on the basis of (1)H- and (13)C-NMR experiments. The new compound (1) was evaluated for its genotoxic and cytotoxic activities and found to possess protective antigenotoxicity in human peripheral blood cells.

[Delta5-7-Ketosterols with modified side chain: the synthesis and the effects on viability and cholesterol biosynthesis in Hep G2 cells].

(22E)-3beta-Hydroxysitosta-5,22-dien-7-one, (22R, 23R)-3beta,22,23-trihydroxysitost-5-en-7-one, and (22R, 23R)-3beta-hydroxy-22,23-isopropylidenedioxysitost-5-en-7-one were synthesized. The cytotoxicity and effects on cholesterol biosynthesis of the resulting 7-ketosterols, 7-ketocholesterol, and (22S,23S)-3beta-hydroxy-22,23-oxidositost-5-en-7-one were studied in hepatoblastoma Hep G2 cells.

Identification of ligands for DAF-12 that govern dauer formation and reproduction in C. elegans.

In response to environmental and dietary cues, the C. elegans orphan nuclear receptor, DAF-12, regulates dauer diapause, reproductive development, fat metabolism, and life span. Despite strong evidence for hormonal control, the identification of the DAF-12 ligand has remained elusive. In this work, we identified two distinct 3-keto-cholestenoic acid metabolites of DAF-9, a cytochrome P450 involved in hormone production, that function as ligands for DAF-12. At nanomolar concentrations, these steroidal ligands (called dafachronic acids) bind and transactivate DAF-12 and rescue the hormone deficiency of daf-9 mutants. Interestingly, DAF-9 has a biochemical activity similar to mammalian CYP27A1 catalyzing addition of a terminal acid to the side chain of sterol metabolites. Together, these results define the first steroid hormones in nematodes as ligands for an invertebrate orphan nuclear receptor and demonstrate that steroidal regulation of reproduction, from biology to molecular mechanism, is conserved from worms to humans.

3-Keto steroids from the marine organisms Dendrophyllia cornigera and Cymodocea nodosa.

The new (20R)-22E-cholesta-4,22-diene-3,6-dione (1), along with three known 3-keto steroids were isolated from the deep-water Mediterranean scleractinian coral Dendrophyllia cornigera (2-4). Moreover, four known related 3-keto steroids were isolated from the sea grass Cymodocea nodosa (5-8). The structure elucidation of steroid 1 and the full NMR resonance assignments of all isolated metabolites were based on interpretation of their spectral data. All compounds are reported for the first time as metabolites of the investigated organisms. Compounds 2 and 3 showed significant cytotoxicity against lung cancer NSCLC-N6 cell line.

Inhibition of 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) activity of human lung microsomes by genistein, daidzein, coumestrol and C(18)-, C(19)- and C(21)-hydroxysteroids and ketosteroids.

Epidemiologic data suggest a relationship between dietary intake of phytochemicals and a lower incidence of some cancers. Modulation of steroid hormone metabolism has been proposed as a basis for this effect. It has been shown that aromatase, 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) are inhibited by the isoflavones, genistein and daidzein, and by coumestrol. In general, the extent of inhibition has been expressed in terms of IC50-values, which do not give information as to the pattern of inhibition, i.e., competitive, non-competitive, or mixed. Less is known of the effects of these compounds on 3alpha-HSD. The human lung is known to have a high level of 17beta-HSD and 3alpha-HSD activity. During the course of studies to characterize both activities in normal and inflamed lung and lung tumors we noted that 3alpha-HSD activity with 5alpha-DHT of microsomes from normal, adult lung was particularly susceptible to inhibition by coumestrol. To clarify the pattern of inhibition, the inhibition constants Ki and K'i were evaluated from plots of 1/v versus [I] and [S]/v versus [I]. Genistein, daidzein and coumestrol gave mixed inhibition patterns versus both 5alpha-DHT and NADH. In contrast, 5alpha-androstane-3,17-dione and 5alpha-pregnane-3,20-dione were competitive with 5alpha-DHT. NAD inhibited competitively with NADH. Our findings demonstrate that phytochemicals have the potential to inhibit 5alpha-DHT metabolism and thereby affect the androgen status of the human lung. The observation of a mixed inhibition pattern suggests these compounds bind to more than one form of the enzyme within the catalytic pathway.

The 11-ketosteroid 11-ketodexamethasone is a glucocorticoid receptor agonist.

Dexamethasone (Dex) is a potent and long-acting glucocorticoid in terms of anti-inflammatory activity without substantial sodium retaining effect. Here, we examine the ability of the 11beta-hydroxyglucocorticoids Dex and cortisol and their 11-keto forms 11-ketodexamethasone (11-ketoDex) and cortisone to bind to glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) and to mediate nuclear translocation and transactivation of a reporter-gene. Unlike cortisone, the 11-ketosteroid 11-ketoDex acts as a potent GR agonist, comparable to Dex and cortisol. Transactivation of MR by Dex or 11-ketoDex was weak or undetectable, despite efficient binding and induction of nuclear translocation. 11beta-HSD2 protects MR and GR from inappropriate occupation by cortisol; it is, however, unable to prevent activation of GR by 11-ketoDex. The finding that 11-ketoDex is a specific GR agonist may explain the potent glucocorticoid effect of Dex in tissues expressing 11beta-HSD2 including kidney and colon and also in certain tumor cells.

New ketosteroids from the red alga Hypnea musciformis.

The dichloromethane/methanol extract from the red alga Hypnea musciformis exhibited PPE elastase inhibition. A diketosteroid, the 20-hydroxy-5alpha-cholest-22-ene-3,6-dione was responsible for this activity. Two new steroids were isolated, 2 was assigned as the 6alpha-hydroxy-cholest-4-ene-3-one and 3 as the 6alpha-hydroxy-cholest-4,22-diene-3-one. The structures were assigned mainly on the basis of 1H and 13C NMR experiments.

Induction of differentiation and apoptosis in human promyelocytic leukemia HL60 cell line by a new type of steroids.

Mer-NF8054X is a new type of steroid whose structure has been established as 11-oxo-18, 22-cycloergosta-6, 8(14)-diene-3beta, 5beta, 9beta, 23S-tetraol (an 18, 22-cycloergostane), which has been reported to have antifungal activity against Aspergillus fumigatus. However, other biological activities are unknown. Herein, we reported that Mer-NF8054X inhibited cell growth of HL60 human leukemia cells, when used either singly or in combination with retinoic acid (RA). In addition, Mer-NF8054X alone induced differentiation and apoptosis of HL60 cells. The induction of differentiation of HL60 cells by Mer-NF8054X was synergistic in combination with RA. On the other hand, Emesterone A, an analogue of Mer-NF8054X which is missing a hydroxy residue from the third position, showed much lower activity than Mer-NF8054X on the inhibition of cell growth and the induction of cell differentiation and apoptosis. However, Emesterone B, an analogue of Emesterone A which is missing a hydroxy residue from the fifth position, showed higher activity than Emesterone A but lower activity than Mer-NF8054X when examined for the inhibition of cell growth and the induction of cell differentiation and apoptosis. These results suggested that Mer-NF8054X and its analogs may be a new type of differentiation inducing agent. The hydroxy residue at the third position or fifth position in Mer-NF8054X may be necessary, but not essential, for inhibition of growth and induction of both differentiation and apoptosis of HL60 cells. In addition, Mer-NF8054X enhanced the differentiation of HL60 cells induced by RA. Based on these results, Mer-NF8054X may have utility in the clinic in combination with RA for leukemia patients.

Evaluation of some 4-fluoro- and 4-cyano derivatives of delta 4,3-ketosteroids as inhibitors of testosterone 5 alpha-reductase.

Some 4-fluorinated analogues of 3-oxo-delta 4 steroids and 4-cyano derivatives of progesterone and androstenedione were evaluated as inhibitors of steroid 5 alpha-reductase activity. Inhibitors of this enzyme may be useful in treating prostatic cancer. 4-Fluoroandrostenedione was a modest inhibitor of the rat enzyme (IC50 = 4.08 microM), while 4-cyanoprogesterone was a potent inhibitor of both the rat and human enzymes (IC50 values = 0.045 microM and 0.050 microM respectively). These two steroids were tested in vivo for activity against androgen sensitive organs in WHT mice. 4-Fluoroandrostenedione caused increases in organ weights, suggesting it is an androgen agonist, while the 4-cyano compound displayed modest androgen ablation. Therefore substitutions at the 4-position may produce compounds of therapeutic use in treating prostate cancer.

[Conformation of the dienone system of delta 4,9-19-nor-3-ketosteroids from x-ray analysis data and its relation to reception and hormonal activity]. / Konformatsiia dienonovoi sistemy delta 4,9-19-nor-3-ketosteroidov po dannym rentgenostrukturnogo analiza i ee sviaz' s retseptsiei i gormonal'noi aktivnost'iu.

Basing on the data of the X-ray analysis of 2 beta-methylestr-4,9-dien-3-one-17 beta-ol (C19H26O2) and A-ring unsubstituted steroid 4,9-dien-3-ones, the noncomplanarity and flexibility of the conjugated dienone system in these transformed steroids has been demonstrated. The results have been also confirmed by UV spectroscopy. The ability of the dienone system to assume conformations with the out of- plane C3 = O3 and/or C9 = C10 bonds allows the AB-fragment of the steroid molecule to adopt the conformations required for interacting with various receptors. This property may also account for a simultaneous enhancement of several hormonal activities upon such a modification of steroids. The results of the X-ray analysis of 2 beta-methylestra-4,9-dien-3-one-17 beta-ol (space group P2(1)2(1)2(1), a 8,543(2), b 9,783(2), c 18,690(7) A, R 8,7%) are presented.

Conformational analysis of 20-keto steroids. Single-crystal X-ray structure analysis of 16 alpha,17-epoxy-4-pregnene-3,20-dione.

The structure of 16 alpha,17-epoxy-4-pregnene-3,20-dione was determined. The 20-carbonyl group eclipses the C(13)-C(17) bond. No direct correlation between the observed structure and its progestational activity could be inferred from our investigation.

[Effect of transformed steroid compounds on mRNA synthesis and glucocorticoid-receptor interaction in thymocytes of adrenalectomized rats]. / Vliianie transformirovannykh steroidnykh soedinenii na sintez mRNK i gliukokortik gliukokortikoidretseptornoe vzaimodeistvie v timotsitakh adrenaléktomirovannykh krys.

Experiments on thymocyte culture of adrenalectomized rats were made to study the effect of 50 transformed steroid compounds containing delta 4-3-keto or delta 5-3-hydroxygroup in ring A, oxygen-containing substituents in ring D and superstructed 20-keto-side chain on glucocorticoid and antiglucocorticoid effects and steroid-receptor interaction. Ten compounds given in a concentration of 10(6) M competed with 3H-triamcinolon for binding thymocyte cytosol on glucocorticoid receptors. Steroid compounds containing the delta 4-3-ketogroup inhibit more actively the incorporation of 3H-uridine into RNA in a concentration of 10(6) M, as compared with compounds containing the delta 5-3-hydroxygroup. Antiglucocorticoid effect on thymocyte function is also produced by 16,17 alpha-isopropylidenedioxy-pregn-5-en-20-on-3 beta-ol; 16,17 alpha-isopropylidenedioxy-pregna-5,21 (21a)-dien-20-on-3 beta-ol at a concentration of 10(6) M.

Studies on the human epididymis: partial characterization of 3 alpha- and 3 beta-hydroxysteroid dehydrogenase, regional distribution of 5 alpha-reductase and inhibitory effect of 4 delta-3-oxosteroids on 5 alpha-reductase.

When [4-14C]-5 alpha-dihydrotestosterone was incubated with the homogenate of human epididymis, 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol were identified as major metabolites. The ratio of 3 alpha- to 3 beta-epimer in androstanediol formation was approximately 2.4. 5 alpha-Androstane-3, 17-dione was also identified as a minor metabolite. Among the subcellular fractions, both the human epididymal 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were localized almost exclusively in the cytosol fraction (105,000 X g supernatant). Both enzymes had optimum pH at 7.5 and optimum temperature at 46 degrees C. NADPH was a more preferable cofactor than NADH for both dehydrogenases. The Michaelis constants (Km) of 3 alpha- and 3 beta-hydroxysteroid dehydrogenase for 5 alpha-dihydrotestosterone were similar and estimated as 8 X 10(-5) M, but the enzymes were unsaturable with the substrate under the conditions investigated, indicating low affinity and high capacity of both dehydrogenases for 5 alpha-dihydrotestosterone. The human epididymal 5 alpha-reductase revealed a regional difference in activity. The 5 alpha-reductase activity in the most proximal part of the head (ductuli efferentes) was one seventh to one tenth the activity in the remaining part of the epididymis which was constructed of ductus epididymis. Except for this finding, the activity of 5 alpha-reductase was highest in the head, then declined along the course to the tail portion. The 5 alpha-reductase for testosterone was competitively inhibited by delta 4-3-oxosteroids such as progesterone, 20 alpha-dihydroprogesterone, 17 alpha-hydroxyprogesterone, 4-androstenedione, 11-deoxycorticosterone, corticosterone and 11-deoxycortisol, which had inhibition constants (Ki) of 3.3 X 10(-9) M, 2.2 X 10(-9) M, 1.8 X 10(-8) M, 1.3 X 10(-8) M, 8.3 X 10(-9) M, 1.5 X 10(-7) M and 8.7 X 10(-8) M, respectively, suggesting the possibility that the 5 alpha-reduction of testosterone is regulated by other delta 4-3-oxosteroids.

Testing transport systems for competition between pairs of reversible inhibitors. Inhibition of erythrocyte glucose transport by cytochalasin B and steroids.

When two different inhibitors, which are unrelated in structure, inhibit transport by binding to the carrier on the same side of the membrane, it is important for an understanding of the mechanism to decide whether they add to a single common site or to two separate sites. To help resolve this problem, kinetic tests are described for determining whether the inhibitors compete with one another. The test involves measuring the inhibition produced by the inhibitors when acting alone or together. The rate observed with both inhibitors present is then compared with that predicted from the separate inhibitions, for the case of either one or two inhibition sites. The test is applied to the inhibition of glucose transport in erythrocytes by cytochalasin B and the steroids androstenedione and androstanedione. These inhibitors were previously shown to bind to sites which are distinct from the substrate site and which are only present in the inward-facing form of the carrier. Cytochalasin B is now found to compete with the steroids. This finding, together with the earlier evidence that both inhibitors sterically protect the carrier against reaction with fluorodinitrobenzene, leads us to conclude that a single site binds both types of inhibitor.

Inhibition of human lymphocyte E-rosette formation by oxygenated sterols.

25-Hydroxycholesterol, 20 alpha-hydroxycholesterol, 7 alpha-hydroxycholesterol, and 5 alpha-hydroxy-6-ketocholestanol, when added to cultures of human lymphocytes in lipoprotein-depleted medium (LPDM) at a concentration of 2.5 x 10(-6) M, inhibit E-rosette formation with sheep red blood cells. 20 alpha-Hydroxycholesterol, 7 alpha-hydroxycholesterol, and 5 alpha-hydroxy-6-ketocholestanol are more potent inhibitors than 25-hydroxycholesterol. The inhibitory effect of 5 alpha-hydroxy-6-ketocholestanol on E-rosette formation appears after 15 min of exposure; with the other three compounds, an exposure time of 18 hr is necessary. The inhibitory effect of E-rosette formation can be abolished by addition of free cholesterol, low-density lipoprotein, or high-density lipoprotein to the LPDM or by incubation of the cells in normal AB serum, but not by the addition of mevalonic acid to the LPDM. These observations suggest that the capacity of oxygenated sterol compounds (OSC) to inhibit E-rosette formation is independent of their inhibitory effect on sterol synthesis. It is possible that OSC inhibit E-rosette formation as a consequence of their insertion into the lymphocyte membrane as cholesterol analogues.