Metabolism of progesterone and estradiol by microsomes and purified cytochrome P-450 RLM3 and RLM5

The metabolism of progesterone and estradiol by cytochrome P-450 RLM3 and RLM5 and by male rat liver microsomes was compared. Products formed were identified by thin layer chromatography and by gas chromatography-mass spectrometry. Eleven metabolites were detected from progesterone metabolism. Three proved to be reduction products formed only by the microsomes and seven metabolites were identified. Those identified were 5 alpha-pregnane-3,20-dione, 3 beta-hydroxy-5 alpha-pregnane-20-one, 2 alpha-hydroxyprogesterone, 6 beta-hydroxyprogesterone, 6 alpha-hydroxyprogesterone, 15-hydroxyprogesterone, and 16 alpha-hydroxyprogesterone. RLM3 formed 6 beta- and 16 alpha-hydroxyprogesterone as major products and 6 alpha- and 15-hydroxyprogesterone as minor ones. RLM5, however, formed 2 alpha-hydroxy- and 16 alpha-hydroxyprogesterone as major products, and 6 beta-hydroxyprogesterone as a minor product. RLM5 and microsomes did not produce detectable levels of 15-hydroxyprogesterone. Two very polar metabolites were produced from progesterone by both RLM3 and RLM5 but were not seen with microsomes and were not identified. Four metabolites were formed from estradiol. One, 15-hydroxyestradiol, was formed only by RLM3. No other product was formed by this enzyme at appreciable rates. RLM5 and microsomes also formed estrone, estriol, and 2-hydroxyestradiol, but the first of these was a very minor product. This study clearly shows a stereospecificity and positional specificity in metabolism of steroid hormones by cytochrome P-450 isozymes of the untreated rat.     

The quantification of endogenous steroids in bovine aqueous humour and vitreous humour using isotope dilution GC-NCI-MS

Pentafluorobenzyloxime-trimethylsilyl derivatives of androgens, progestogens and corticosteroids were prepared and used for the analysis of these steroids in bovine aqueous humour and vitreous humour by GC-MS method. Appropriate deuteriated isotopomers of the parent steroids were labelled with deuterium via simple synthetic procedure and used as internal standards. The concentration (ng ml(-1), +/- S.E.M.) of these steroids in bovine aqueous humour and vitreous humour were found to be as follow: (1) aqueous humour (n = 17); hydrocortisone (n = 17; 2.40 +/- 0.54), progesterone (n = 15; 0.06 +/- 0.01), 4-androstene-3,17-dione (n = 8; 0.15 +/- 0.07) and testosterone (n = 4; 0.14 +/- 0.04); and (2) bovine vitreous humour (n = 19); hydrocortisone (n = 19; 1.78 +/- 0.25), progesterone (n = 18; 0.09 +/- 0.01), 4-androstene-3,17-dione (n = 19; 0.11 +/- 0.02), 11-deoxycorticosterone (n = 12; 29.27 +/- 6.42), 17alpha-hydroxyprogesterone (n = 6; 5.55 +/- 3.12). The concentration of corticosterone, 11-deoxycorticosterone and 17alpha-hydroxyprogesterone and testosterone and corticosterone were below the limit of detection in aqueous humour and vitreous humour, respectively.     

Structure-activity relationship of various corticosteroids on the feedback control of corticotrophin secretion.

Several steroids occurring in the pathway of corticosteroid biosynthesis were investigated for their ability to exert a fast or delayed feedback inhibition of stress-induced release of corticotrophin. Rats were injected subcutaneously with vehicle or a steroid either 10 min (fast feedback) or 4 h (delayed feedback) before they were subjected to stress which consisted of a 2 min exposure to ether vapour. 2 Changes in plasma corticosterone concentration and in vitro corticosterone production by excised adrenal glands were used as indices of corticotrophin release. 3 Among the steroids tested only 11beta, 21-dihydroxypregn-4-ene-3, 20-dione (corticosterone) and 11beta, 17alpha, 21-trihydroxypregn-4-ene-3, 20-dione (cortisol) inhibited the stress response 10 min after their administration. Therefore, it appears that the fast feedback mechanism is limited to steroids with a 21-hydroxyl and a 11beta-hydroxyl group. 4 In contrast, many steroids caused inhibition of the stress response 4 h after their administration. These steroids were corticosterone, cortisol, 21-hydroxypregn-4-ene-3, 20-dione (11-deoxycorticosterone), 17alpha, 21-dihydroxypregn-4-ene-3, 20-dione (11-deoxycortisol), 11beta-hydroxypregn-4-ene-3, 20-dione (11beta-hydroxyprogesterone) and 11beta, 17alpha-dihydroxypregn-4-ene-3, 20-dione (11beta, 17alpha-dihydroxyprogesterone). Thus, either the 21-hydroxyl group (e.g. 11-deoxycorticosterone) or the 11beta-hydroxyl group (e.g. 11beta-hydroxyprogesterone) is sufficient for delayed feedback activity. The 11alpha-hydroxyl group, e.g. 11alpha, 17alpha, 21-trihydroxypregn-4-ene-3, 20-dione (11-epicortisol) renders the steroid inactive on both feedback mechanisms. 5 18,21-Dihydroxypregn-4-ene-3, 20-dione (18-hydroxydeoxycorticosterone) was found to be the only steroid that is secreted by the adrenal gland of the rat in quantities sufficient to cause exaggeration of the stress-induced release of corticotrophin. This steroid has been implicated as a possible hypertensive agent, and its role in the control of corticotrophin secretion is discussed here.     

Ketoconazole inhibition of the bifunctional cytochrome P450c17 does not affect androgen formation from the endogenous lyase substrate. The catalytic site remains refractory in the course of intermediary hydroxyprogesterone processing.

The inhibition of the bifunctional steroidogenic cytochrome P450c17 (CYP17: steroid-17 alpha-hydroxylase/steroid-17,20-lyase) by the imidazole-type fungicide, [(+/-)-cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl- methyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine) (ketoconazole), was investigated with the aim of differentiating between effects on androgen formation from exogenously added and endogenously produced 17 alpha-hydroxyprogesterone. Using microsomal membranes from rat testis, turnover of progesterone by P450c17 was competitively inhibited by ketoconazole with KI = 0.40 microM. Ketoconazole did not affect the linear relationship between the ratio of productive events (corresponding to androgen formation rates) versus abortive events (corresponding to 17 alpha-hydroxyprogesterone formation rates) and the sum of catalytic events. This was an indication that this inhibitor did not interfere with intermediate processing by P450c17. Androgen formation from exogenous but not from endogenous 17 alpha-hydroxyprogesterone was competitively inhibited by ketoconazole. The simultaneous conversion of 1 microM each of [3H]progesterone and 17 alpha-hydroxy[14C]progesterone was also reduced by ketoconazole. Calculation of 3H/14C ratios in the 17 alpha-hydroxyprogesterone and androgen fractions revealed that the endogenous 17 alpha-hydroxyprogesterone pool was metabolized to androgens at rates 6.4, 11.6, 17.6 and 21.2-fold faster than the exogenous pool in the presence of 0.5, 1, 2 and 4 microM ketoconazole, respectively; this value was only 4.0 in controls. It is concluded that ketoconazole inhibits turnover of steroid ligands only when they approach the P450c17 active site in a substrate-state and that inhibition of androgen formation from progesterone is due to inhibition of the first catalytic step only. A model is described in which the P450c17 active site is refractory towards ketoconazole when the intermediary steroid is retained and being processed at that site.     

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.     

Inhibition of the effects of alfathesin and other steroid anesthetics by catatoxic steroids in rats.

In rats, the sleeping time induced by overdosage with eight steroid anesthetics--alfathesin, 3-(3-oxo-17beta-hydroxy-19-nor-4-androsten-17alpha-yl)-propionic acid-lactone (SC-8109), 21-hydroxy=5alpha-pregnane-3,20-dione (P-234), 4-pregnene-3,11,20-trione (Bio.66), 17-hydroxy-3-oxo-4-androstene-17alpha-propionic acid-gamma-lactone(SC-5233),3alpha-hydroxy-5beta-pregnane-11,20-dione, 5beta-pregnane-3,11,20-trione (U-1373), and hydroxydione--was abolished or considerably reduced by a variety of catatoxic compounds, particularly 3beta-hydroxy-20-oxo-5-pregnene-16alpha-carbonitrile (PCN), 9alpha-fluoro-11beta,17-dihydroxy-3-oxo-4-androstene-17alpha-propionic acid potassium salt (CS-1), prednisolone, ethylestrenol and spironolactone. Phenobarbital and diphenylhydantoin, two non-steroidal stimulators of hepatic microsomal drug metabolism, were also highly effective. In contrast, triamcinolone, estradiol,progesterone, desoxycorticosterone and hydroxydione, which exert little or no catatoxic activity, failed to significantly diminish anesthesia or sedation.     

Over-the-counter delta5 anabolic steroids 5-androsen-3,17-dione; 5-androsten-3beta, 17beta-diol; dehydroepiandrosterone; and 19-nor-5-androsten-3,17-dione: excretion studies in men

Studies of urinary steroids were performed in males after oral administration of 5-androsten-3,17-dione; 5-androsten-3beta,17beta-diol; dehydroepiandrosterone; and 19-nor-5-androsten-3,17-dione. 5-Androsten-3,17-dione; 5-androsten-3beta,17beta-diol; and dehydroepiandrosterone amplify most endogenous steroids, but to a lesser extent than their delta4 analogues do. Especially affected are androsterone, etiocholanolone, dehydroandrosterone, dehydroepiandrosterone, and isomeric 5-androstendiols. 5-Androsten-3,17-dione; 5-androsten-3beta,17beta-diol; and dehydroepiandrosterone elevate the urinary testosterone to epitestosterone (T/E) ratio by a factor of 2-3 a few hours after administration. This may cause a positive T/E test (> 6) for individuals with normal T/E ratios higher than 2. Most of the steroids return to their original concentrations in less than 24 h. Etiocholanolone and 5beta-androstan-3alpha,17beta-diol remain elevated for several days. A reduced androsterone to etiocholanolone (A/E) ratio may be an indication of delta5 steroids abuse. 19-Nor-5-androsten-3,17-dione has a similar effect, except that all metabolites in urine are 19-nor exogenous steroids. Identification criteria for 19-nor-5-androsten-3,17-dione may be the same as nandrolone, that is, detection of 19-norandrosterone and 19-noretiocholanolone. Specific abundant metabolites of 19-nor-5-androsten-3,17-dione are 19-nordehydroandrosterone and 19-nordehydroepiandrosterone. In the later stages of excretion, higher concentration of 1 9-noreticholanolone relative to 19-norandrosterone specifically indicates administration of 19-nor delta5 steroids.     

Changes in steroid hormone metabolism in rat liver microsomes following administration of 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD).

Treatment of male and female rats with 4 × 20 μg/kg of 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD) led to 2–3-fold increases in liver microsomal cytochrome P450 levels concomitantly with changes in liver microsomal metabolism of 4-androstene-3,17-dione, 5α-androstane-3α,17β-diol and 4-pregnene-3, 20-dione. The changes were most pronounced in female rats where some hydroxylase activities increased 3–5-fold. It is suggested that the observed effects following TCDD administration may be related to the endocrine symptoms sometimes seen in patients exposed to this drug.     

Steroid synthesis in ovarian homogenates from hypophysectomized adult female mice treated with diethylstilbestrol in neonatal life.

Eight-week-old female NMRI-mice treated with 5 micrograms diethylstilbestrol (DES) or vehicle (olive oil) from d 1 to 5 after birth were hypophysectomized or sham operated. Thirty days after the operation, homogenates were prepared from the ovaries and incubated at 37 degrees C in the presence of [3H]pregnenolone for 1 h. In parallel experiments ovaries were taken for histology. In control ovaries regressing corpora lutea were still present 4 wk after hypophysectomy (Hx). Ovaries from DES females had no corpora lutea, but showed degeneration of the pre-Hx hypertrophic interstitial tissue. Steroids synthesized during the incubation were extracted and separated in a two-dimensional thin-layer chromatography system. After Hx, the amount of extractable [3H]testosterone, [3H]progesterone, and [3H]androstenedione recovered from homogenates of vehicle-treated females increased, but the amount of recovered [3H]-17 alpha-hydroxyprogesterone was not significantly altered. In contrast, in DES-treated females Hx significantly decreased levels of [3H]androstenedione and [3H]-17 alpha-hydroxyprogesterone, but did not affect [3H]progesterone or [3H]testosterone. The persistent difference in steroid synthetic activity after Hx could be linked to the difference in morphology between different ovarian tissue compartments of control and DES-treated females.     

Development of a radioreceptor assay to measure glucocorticoids.

We have developed a radioreceptor assay to measure glucocorticoids. The assay employs the partially purified 95-kDa receptor isolated from human liver and purified by size fractionation on high-performance liquid chromatography (HPLC). In the assay [3H]prednisolone competes with steroids (endogenous and exogenous) for binding to the receptor. Bound and free are separated by treatment with charcoal. The between-day precision [% coefficient of variation (CV)] at concentrations of 9.4, 18.7, and 69.9 micrograms/L prednisolone is 16.6, 9.3 and 4.5%, respectively. Specificity studies revealed that hydrocortisone, deoxycorticosterone, 4-pregnene-17 alpha,21-diol-3,20-dione, 17 alpha-hydroxyprogesterone, corticosterone and beta-hydroxyprogesterone all compete with [3H]prednisolone for binding to the receptor. Prednisone and 6 alpha-methyl prednisolone displace [3H]prednisolone to only a minor degree. The assay has been used to assess "glucocorticoid activity" in children with rheumatic diseases treated with prednisolone.     

RECEPTORS AND STEROID-DEPENDENT HYPERTENSION

1. Repeated observations indicate that ACTH administration causes hypertension. 2. Development of hypertension requires 17 alpha-hydroxyprogesterone and 17 alpha,20 alpha-dihydroxy-4-pregnene-3-one to be present in association with other steroids. 3. The hypertensinogenic activity of corticosteroids is distinct from their glucocorticoid and mineralocorticoid effects. 4. The location of central and peripheral receptors for this hypertensinogenic activity is not clear. 5. The physiological mechanisms that mediate the response are unknown, though a number of potential mediating effects has been demonstrated. 6. The overall importance of unusual steroids and steroid actions in human essential hypertension still requires elucidation.     

Progesterone metabolism in recombinant yeast simultaneously expressing bovine cytochromes P450c17 (CYP17A1) and P450c21 (CYP21B1) and yeast NADPH-P450 oxidoreductase.

Simultaneous expression plasmids were constructed for bovine adrenal cytochromes P450c17 and P450c21 (pA gamma alpha) and for both P450s together with NADPH-cytochrome P450 reductase (pAR gamma alpha). On introduction of each of the plasmids into Saccharomyces cerevisiae AH22 cells, the transformed yeast strains AH22/pA gamma alpha and AH22/pAR gamma alpha produced about 10(5) molecules per cell of P450c17 and 2 x 10(3) molecules per cell of P450c21. The expression levels of NADPH-cytochrome P450 reductase was about 3 x 10(4) and 6 x 10(5) molecules per cell in the strains AH22/pA gamma alpha and AH22/pAR gamma alpha, respectively. When progesterone was added to growing cell cultures of the transformed yeast strains, the substrate was metabolized more rapidly in the AH22/pAR gamma alpha cells than AH22/pA gamma alpha cells, probably due to overproduction of the reductase. In the AH22/pAR gamma alpha cells, progesterone was first converted into 17 alpha-hydroxyprogesterone to the extent of 82% by the catalysis of P450c17. 17 alpha-hydroxyprogesterone was further converted into 11-deoxycortisol by P450c21 to the extent of 60% of the added substrate. The conversion of progesterone into androstenedione through 17 alpha-hydroxyprogesterone was estimated to be less than 3%, suggesting very low C17,20-lyase activity of P450c17, although other hydroxylation products were detected. Androstenedione was further converted into testosterone by an unknown pathway present in S. cerevisiae cells.     

Characterization of related impurities in megestrol acetate

Three new compounds, 17alpha-acetoxy-2,6-dimethylpregna-1,4,6-triene-3,20-dione (1), 17alpha-acetoxy-2alpha,6-dimethylpregna-4,6-diene-3,20-dione (2), 17alpha-acetoxy-6alpha-methoxylmethylpregna-4-ene-3,20-dione (3), together with five known ones, 17alpha-acetoxy-6beta-hydroxyl-6alpha-methylpregna-4-ene-3,20-dione (4), 17alpha-acetoxy-6alpha-hydroxyl-6beta-methylpregna-4-ene-3,20-dione (5), 17alpha-acetoxy-pregna-4-ene-3,6,20-trione (6), 17alpha-acetoxy-pregna-4-ene-3,20-dione (7) and 17alpha-acetoxy-6-methylene-pregna-4-ene-3,20-dione (8), were isolated and identified from the residual mother liquor of megestrol acetate. Their structures were established by spectroscopic methods. These compounds seem to be minor impurities in production of the drug megestrol acetate.     

Effects of various pregnanes and two 23-nor-5-cholenic acids on cardenolide accumulation in cell and organ cultures of Digitalis lanata

5-Pregnen-3beta-ol-20-one (pregnenolone), 4-pregnene-3,20-dione (progesterone), 5-pregnene-3beta,21-diol-20-one (21-hydroxypregnenolone), 4-pregnen-21 -ol-3,20-dione (cortexone), 5beta-pregnane-3,20-dione, 5alpha-pregnane-3,20-dione, 5beta-pregnan-3alpha-ol-20-one, 5beta-pregnan-3beta-ol-20-one, 5beta-pregnane-3beta,14beta, 21-triol-20-one 3-acetate, 23-nor-5-cholenic acid-3beta,20xi-diol, and 23-nor-3,20(22) E-choladienic acid-3beta-ol were administered to photomixotrophic shoot cultures of Digitalis lanata Ehrh. capable of synthesizing cardenolides, as well as to cardenolide-free tissue cultures, such as auxotrophic, dark-grown shoot cultures and cell suspension cultures of the same plant species. None of the pregnane precursors was qualified to restore cardenolide biosynthesis in the cardenolide-free tissues. The cardenolide content of light-grown shoot cultures, on the other hand, increased by 161%, 240%, 30%, 430% and 80% when 100 mg l(-1) of 21-hydroxypregnenolone, 5beta-pregnane-3,20-dione, 5beta-pregnan-3beta-ol-20-one, 5beta-pregnane-3beta,14beta,21-triol-20-one, 23-nor-5,20 (22) E-choladienic acid-3beta-ol, respectively, were administered. Pregnenolone, progesterone, cortexone, 5alpha-pregnanes, 5beta -pregnan-21-ols, and 23-nor-5-cholenic acid-3beta,20xi-diol, on the other hand, had no visible effect. Two different types of cardenolides (termed fucose-type cardenolides and digitoxose-type cardenolides) were identified which may be formed via different biosynthetic routes. The "norcholanic acid pathway" seems to be operative in D. lanata shoot cultures only in the formation of fucose-type cardenolides.     

Amnesia-reversal activity of a series of cyclic imides

A series of dihydro-1H-pyrrolizine-3,5(2H,6H)-diones were synthesized and evaluated for their ability to reverse electroconvulsive shock (ECS) induced amnesia in mice. Among the structure-activity relationships explored were the effects of ring size, the presence of heteroatoms (sulfur) in the ring system, and the introduction of alkyl substituents. The optimal ring size for the bicyclic system was 5.5 with dihydro-1H-pyrrolizine-3,5(2H,6H)-dione (3), although some activity was present in the corresponding 5.6 [hexahydro-3,5-indolizinedione (7)] and 6.6 [tetrahydro-2H-quinolizine-4,6(3H,7H)-dione (9)] analogues. Replacement of the C-1 carbon atom in compound 3 with a sulfur [dihydropyrrolo[2,1-b]thiazole-3,5(2H,6H)-dione (10)] abolished activity, and the introduction of methyl groups resulted in poorer biological profiles except when the substitution was made at the 7a position [dihydro-7a-methyl-1H-pyrrolizine-3,5(2H,6H)-dione (4)]. In several instances, hydrolysis of the parent bicyclic compound was carried out to furnish the corresponding lactam acids, which were further derivatized. Several exhibited interesting activity, especially the 5-oxo-2-pyrrolidinepropanoic acid derivatives such as 5-oxo-2-pyrrolidinepropanoic acid (12), 5-oxo-2-pyrrolidinepropanoic acid phenylmethyl ester (17), 5-oxo-2-pyrrolidinepropanoic acid (3-chlorophenyl)methyl ester (20), N-4-pyridyl-5-oxo-2-pyrrolidinepropanoic acid amide (25), and N-(2,6-dimethylphenyl)-5-oxo-2-pyrrolidinepropanoic acid amide (27). Compound 3 (CI-911; rolziracetam) was also observed to improve performance on a delayed-response task in aged rhesus monkeys and was selected for evaluation in cognitively impaired human subjects on the basis of its biological profile and a wide margin of safety in animals.     

甾体激素 Ⅱ.9α-氟副肾皮质激素的新合成路线

自16α,17α-环氧孕甾-4-烯-11α-醇-3,20-二酮(Ⅰ)經对甲苯磺酰化和消去反应得到16α,17α-坏氧孕甾-4,9(11)-二烯-3,20-二酮(Ⅱ),用氫溴酸打开环氧。氫解脫溴得到孕甾-4,9(11)-二烯-17α-醇-3,20-二酮(Ⅲ),引入C₂₁-OAc得到孕甾-4,9(11)-二烯-17α,21-二醇-3,20-二酮21-醋酸脂(Ⅳ),然后按已知方法合成9α-氟-可的唑(Ⅵ).(Ⅵ)对(Ⅰ)的收率为17.4%理論。也进行了由(Ⅲ)先引入9β,11β-环氧,然后引入C₂₁-OAc以合成9α-氟可的唑的試探。     

Bromine- and iodine-substituted 16alpha,17alpha-dioxolane progestins for breast tumor imaging and radiotherapy: synthesis and receptor binding affinity

Progesterone receptors (PRs) are present in many breast tumors, and their levels are increased by certain endocrine therapies. We describe the synthesis and PR binding affinities of a series of bromine- and iodine-substituted 16alpha,17alpha-dioxolane progestins, some of which, when appropriately radiolabeled, are potential agents for diagnostic imaging of PR-positive breast tumors using positron emission tomography (PET) and for radiotherapy. These compounds were synthesized from halogenated furanyl, phenyl, and thiophenyl aldehydes and a progestin 16alpha,17alpha,21-triol (5) in the presence of HClO4 or Sc(OTf)3 in high yields under optimized conditions. A new reagent, perfluoro-1-butanesulfonyl fluoride (PBSF), was used to convert the C-21 OH to F in high yields. The relative binding affinities (RBAs) of the most promising compounds for the PR (RBA of R5020 = 100) were 16alpha,17alpha-[(R)-1'-alpha-(5-bromofurylmethylidene)dioxyl]-21-hydroxy-19-norpregn-4-ene-3,20-dione (endo-6; RBA = 65 and moderate lipophilicity), 21-fluoro-16alpha,17alpha-[(R)-1'-alpha-(5-iodofurylmethylidene)dioxyl]-19-norpregn-4-ene-3,20-dione (endo-14; RBA = 40) and 21-fluoro-16alpha,17alpha-[(S)-1'-beta-(4-iodophenylmethylidene)dioxyl]-19-norpregn-4-ene-3,20-dione (exo-16; RBA = 34).     

穆库没药甾体类化学成分的研究

目的:对穆库没药甾体类化学成分进行系统研究.方法:采用硅胶柱色谱、ODS柱色谱及制备HPLC等方法分离纯化,运用现代光谱学方法鉴定化合物结构.结果:从穆库没药中分离得到5个化合物,分别鉴定为4,17(20)-(cis)-pregnadiene-3,16-dione (1)、progesterone (2)、20-acetyloxy-4- pregnene-3,16-dione (3)、Z-4,17(20)-pregnadiene-3,16-dione (Z-Guggulsterone)(4)、E-4,17(20)- pregnadiene-3,16-dione (E-Guggulsterone)(5).结论:化合物1-2从穆库没药中首次分离得到.     

Synthesis and in vitro anticancer activity of novel thiazacridine derivatives

Acridine derivatives represent a well-known class of anticancer agents that generally interfere with DNA synthesis and inhibit topoisomerase II. A series of eight new 3-acridin-9-ylmethyl-thiazolidine-2,4-dione and 3-acridin-9-ylmethyl-5-arylidene-thiazolidine-2,4-dione derivatives were synthesized. All the compounds were evaluated for their cell antiproliferation activity with the 3-(4,5-dimethyl-2-thiozolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT assay. The antiproliferative effects of the synthesized compounds were tested against several tumoral cell lines, namely SF-295 (central nervous system), HCT-8 (colon carcinoma), and MDA-MB-435 (melanoma) cells using doxorubicin as a positive control. Among the synthesized compounds, 3-acridin-9-ylmethyl-5-acridin-9-ylmethylene-thiazolidine-2,4-dione, 3-acridin-9-ylmethyl-5-(4-methoxy-benzylidene)-thiazolidine-2,4-dione, and 3-acridin-9-ylmethyl-5-(4-bromo-benzylidene)-thiazolidine-2,4-dione exhibited the most potent anticancer activity against the HCT-8 and MDA-MB-435 cell lines. After a detailed analysis of the structure of the thiazacridine molecules, we revealed the main possible interactions using the compound 3-acridin-9-ylmethyl-5-acridin-9-ylmethylene-thiazolidine-2,4-dione as an example. The benefits of these compounds, regardless of the pharmacological target are the presence of two aromatic rings (pi systems), significant planarity (intercalating ability) and the presence of three hydrogen-bond acceptors, two of which are stronger (oxygen atoms) than the other (sulfur atom).     

11beta-hydroxyprogesterone acts as a mineralocorticoid agonist in stimulating Na+ absorption in mammalian principal cortical collecting duct cells

The binding of mineralocorticoid hormones to the mineralocorticoid receptor is the first step in a cascade of events leading to the stimulation of Na(+) reabsorption by renal cortical collecting duct (CCD) principal cells. The agonist properties of mineralocorticoid hormones are linked to contacts between their 21-hydroxyl group and Asn770, a residue of the ligand-binding domain of the human mineralocorticoid receptor (hMR). Here, we investigate whether the presence of a hydroxyl group at position 11, 17, or 20 could also alter the activity of progesterone (P), a mineralocorticoid antagonist without the 21-hydroxyl group. Both 17 alpha-hydroxyprogesterone (17OHP) and 20 alpha-hydroxyprogesterone (20OHP) antagonized the aldosterone-induced trans-activation activity (IC(50): 17OHP, 10(-7) M; 20OHP, 10(-8) M) of the hMR transiently expressed in COS-7 cells lacking steroid receptors. In cultured mouse mpkCCD(cl4) principal cells, 17OHP and 20OHP also prevented the aldosterone-stimulated amiloride-sensitive component of the short-circuit current (Ams I(sc)), reflecting Na(+) absorption mediated by the epithelial Na(+) channel (ENaC). In contrast, 11 beta-hydroxyprogesterone (11OHP) activated the transiently expressed hMR in COS-7 cells in a dose-dependent manner (ED(50): 10(-8) M) and, like aldosterone, stimulated Ams I(sc) in mpkCCD(cl4) cells. Docking 11OHP within the hMR-ligand-binding domain homology model revealed that the agonist activity of 11OHP is caused by contacts between its 11 beta-hydroxyl group and Asn770. Furthermore, 11OHP was unable to activate the mutant hMR/N770A, in which Ala is substituted for Asn at position 770. These findings demonstrate that in the absence of the 21-hydroxyl group, the 11 beta-hydroxyl group can produce the contact with the hMR-Asn770 required for the hMR activation leading to stimulated Na(+) absorption.