Metabolism of the cholestanol precursor cholesta-4,6-dien-3-one in different tissues

Cerebrotendinous xanthomatosis (CTX) is a lipid storage disease where the basic defect is a lack of the mitochondrial C27-steroid 26-hydroxylase involved in bile acid synthesis (EC 1.14.13.15). Cholestanol and cholesterol accumulate in all tissues. At least part of the accumulation of cholestanol is due to a 7 alpha-dehydroxylation of early bile acid intermediates. Cholesta-4,6-dien-3-one, a proposed intermediate in this pathway, is found in increased concentrations in serum of the patients. This study shows that cholesta-4,6-dien-3-one may be metabolized to 4-cholesten-3-one and cholestanol by liver, adrenals and brain. No conversion was found in intestinal mucosa or in kidneys. The capacity to convert cholesta-4,6-dien-3-one into 4-cholesten-3-one and cholestanol varied in different tissues as well as in different species. The results are discussed in relation to the cholestanol accumulation in CTX.     

3 beta-hydroxyandrost-4-en-6-one derivatives as aromatase inhibitors

The 3-formate (II), 3-acetate (III), 3-bromoacetate (IV), 3-propionate (V), 3-methyl ether (VI), and 3-deoxy-derivative (VII) of 3 beta-hydroxyandrost-4-ene-6,17-dione (I) were synthesized and tested in human placental microsomes for their ability to inhibit aromatase. II, III, and VII of this series were potent inhibitors of aromatase with the IC50's (1.7 and 3.3 microM) of the latter two comparable to that (1.2 microM) of 4-hydroxyandrostenedione. Kinetic studies showed that the three steroids are competitive inhibitors of the enzyme with Ki's of 16.0, 5.5, and 0.61 microM for II, III, and VII. Furthermore, II showed a time-dependent, pseudo-first order rate of inactivation of aromatase with Ki of 20.5 microM and kinact of 1.54 x 10(-2) min-1, while III gave a time-dependent, biphasic loss of the enzyme activity. NADPH and oxygen were required for the time-dependent inactivation and the substrate, androstenedione, prevented it.     

The identification of 14 alpha,17 beta-dihydroxyandrost-4-en-3-one monohydrate and 14 alpha,17 beta-dihydroxyandrosta-1,4-dien-3-one monohydrate, metabolites of androstenedione in Mucor piriformis.

The microorganism Mucor piriformis transforms androst-4-ene-3,17-dione into a major and several minor metabolites. X-ray crystallographic analysis of two of these metabolites was undertaken to determine unambiguously their composition and chirality. Crystals belong to the orthorhombic space-group P2(1)2(1)2(1), with a = 7.199(4) A and a = 6.023(3) A, b = 11.719(3) A and b = 13.455(4) A, c = 20.409(3) A and c = 20.702(4) A for the two title compounds, respectively. The structures have been refined to final R values of 0.060 and 0.040, respectively.     

Mechanism and stereochemistry in the sequential enzymatic saturation of the two double bonds in cholesta-4,6-dien-3-one.

The mechanism and stereochemistry in connection with enzymatic conversion of cholesta-4,6-dien-3-one into cholestanol was studied. Rat and mouse liver microsomes are able to catalyze NADPH-dependent sequential saturation of the two double bonds. Evidence was obtained that the saturation of the delta 6-double bond includes transfer of a hydride ion from the B-side of the cofactor to the 7-position of the steroid (mainly 7 beta-position), followed by addition of a proton to the 6 alpha-position (mainly trans addition). The saturation of the delta 4-double bond includes transfer of a hydride ion from the B-side of the cofactor to the 5 alpha-position of the steroid followed by addition of a proton to the 4 beta-position (trans addition). The reduction of the 3-oxo group was found to involve transfer of a hydride ion from the B-side of the cofactor NADPH to the 3 alpha-position of the steroid. The results are in accord with the contention that the enzymatic saturation of the two double bonds involves a polarization of the 3-oxo group making C-7 electrophilic and C-6 nucleophilic in connection with the saturation of the delta 6-double bond and C-5 electrophilic and C-4 nucleophilic in connection with the saturation of the delta 4-double bond.     

26-Hydroxycholesterol and cholest-4-en-3-one,the first metabolites of cholesterol in potato plants

Etiolated potato sprouts convert administered cholesterol-4-¹⁴C to radioactive 26-hydroxycholesterol and cholest-4-en-3-one. These two steroids must be the first products of cholesterol metabolism in potato plants.     

The role of a cholesta-8,14-dien-3β-ol system in cholesterol biosynthesis

The biosynthesis of cholesterol from squalene and tritiated water is described. Degradation of the cholesterol indicated that C-15 may be involved in cholesterol biosynthesis. In accordance with this view it is shown that in the conversion of [2RS-(3)H(2)]mevalonic acid into cholesterol one of the hydrogen atoms at C-15 is removed. A mechanism for the removal of the 14alpha-methyl group in steroid biosynthesis that involves the labilization of a C-15 hydrogen atom is outlined. In accordance with the requirement of this scheme it is shown that 4,4'-dimethyl-cholesta-8,14-dien-3beta-ol is converted into cholesterol.     

Cytochrome P-450scc-catalyzed production of progesterone from 22R-hydroxycholest-4-en-3-one by way of 20,22-dihydroxycholest-4-en-3-one

Transient accumulation of a dihydroxylated steroid was found when 22R-hydroxycholest-4-en-3-one was used as the substrate for a reconstituted cholesterol side-chain cleavage system derived from bovine adrenocortical mitochondria. The indications were that the accumulated steroid was an intermediate in the cytochrome P-450scc-catalyzed reaction. The retention time of the accumulated intermediate was identical with that of authentic 20,22-dihydroxycholest-4-en-3-one on HPLC. When 22R-hydroxycholesterol and 22R-hydroxycholest-4-en-3-one were incubated simultaneously, the total amount of reaction products was essentially the same as that observed with 22R-hydroxycholest-4-en-3-one alone. Under the conditions employed, the apparent turnover number of cytochrome P-450scc for 22R-hydroxycholesterol was calculated to be 77 nmol/min/nmol P-450 from the amount of pregnenolone formed, whereas the apparent turnover number for 22R-hydroxycholest-4-en-3-one was 64 nmol/min/nmol P-450 with respect to the intermediate formation and 77 nmol/min/nmol P-450 with respect to the progesterone formation. The apparent turnover number for 20,22-dihydroxycholest-4-en-3-one was about 125 nmol/min/nmol P-450, which was not significantly different from that of 20,22-dihydroxycholesterol. The apparent Km for 22R-hydroxycholesterol was about 20 microM and those for 22R-hydroxycholest-4-en-3-one and 20,22-dihydroxycholest-4-en-3-one were 50 and 40 microM, respectively. Thus, 22R-hydroxycholest-4-en-3-one was efficiently metabolized to progesterone by way of 20,22-dihydroxycholest-4-en-3-one by cytochrome P-450scc.     

Nostoc muscorum: a regioselective biocatalyst for 17-carbonyl reduction of androst-4-en-3,17-dione and androst-1,4-dien-3,17-dione

Nostoc muscorum PTCC 1636 was examined for its ability to convert androst-4-en-3,17-dione (AD) and androst-1,4-dien-3,17-dione (ADD) to their 17-hydroxy related derivatives in BG-11 medium. Bioconversion procedures were carried out at 25 °C without shaking. The metabolites obtained were purified using chromatographic methods and characterized as testosterone and 1-dehydrotestosterone on the basis of their spectroscopic features. In both cases, the bioreaction characteristics observed were 17-carbonyl reduction.     

Synthesis of 3 beta-hydroxy-5 alpha-cholest-8-en-7-one and 3 beta-hydroxy-5 alpha-cholest-8-en-11-one: evaluation as potential hypocholesterolemic agents

An efficient procedure for the chemical synthesis of 3 beta-hydroxy-5 alpha-cholest-8-en-7-one and 3 beta-hydroxy-5 alpha-cholest-8-en-11-one is described. These ketosterols have been shown to have possible significant hypocholesterolemic effects when fed to normal rats at a level of 0.15% in a laboratory chow diet. The diets containing the steroids caused significant decreases in food consumption which were associated with decreases in the rate of gain in body weight.     

Enzyme-assisted synthesis and structure characterization of glucuronide conjugates of methyltestosterone (17 alpha-methylandrost-4-en-17 beta-ol-3-one) and nandrolone (estr-4-en-17 beta-ol-3-one) metabolites

A new and useful method based on enzyme-assisted synthesis was developed for producing 3 alpha-O-beta-D-glucuronide conjugates from synthetic phase I metabolites of methyltestosterone and nandrolone. The formed glucuronide conjugates of 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol (I), 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (II), 5 alpha-estran-3 alpha-ol-17-one (III), and 5 beta-estran-3 alpha-ol-17-one (IV) are urinary metabolites, indicating the human misuse of the above-mentioned anabolic androgenic steroids (AAS). The common lack of reference material precludes the use and validation of these biomarkers in human doping control. Liver microsomes from Aroclor 1254-induced rats were used as a highly active source of mammalian UDP-glucuronosyltransferases (UGT, EC 2.4.1.17). After purification by protein precipitation, liquid-liquid extraction (dichloromethane), C-18 solid-phase extraction, and lyophilization, the steroid glucuronide structures were characterized by (1)H and (13)C NMR spectroscopy and tandem mass spectrometry. The enzymatic method was highly stereoselective, producing a single major conjugate from the parent steroids I-IV. The stereochemically pure steroid glucuronide conjugates were recovered in milligram amounts (1.0-2.8 mg, yield 12-29%), which is sufficient for veterinary and human doping control analyses; for pharmaco-, toxico-, and enzyme kinetic studies in the pharmaceutical industry; for clinical laboratories; and for forensic medicine. A new sensitive LC-MS method was developed for controlling the product purity in syntheses, as well as for enzyme kinetic characterization of AAS-metabolizing UGT activities in rat liver toward the aglycones I-IV. In this study, the UGT enzymes responsible for the formation of 3 alpha-O-linked glucuronides from the substrates I, II, III, and IV exhibited the specific enzyme activity values: 25, 124, 48, and 212 nmol/mg microsomal protein in a 2-h incubation, respectively.     

Microbial transformation of cadina-4,10(15)-dien-3-one, aromadendr-1(10)-en-9-one and methyl ursolate by Mucor plumbeus ATCC 4740

The sesquiterpenes cadina-4,10(15)-dien-3-one (1) and aromadendr-1(10)-en-9-one (squamulosone) (14) along with the triterpenoid methyl ursolate (21) were incubated with the fungus Mucor plumbeus ATCC 4740. Substrates 1, 14 and ursolic acid (20) were isolated from the plant Hyptis verticillata in large quantities. M. plumbeus hydroxylated 1 at C-12 and C-14. When the iron content of the medium was reduced, however, hydroxylation at these positions was also accompanied by epoxidation of the exocyclic double bond. In total nine new oxygenated cadinanes have been obtained. Sesquiterpene 14 was converted to the novel 2alpha,13-dihydroxy derivative along with four other metabolites. Methyl ursolate (21) was transformed to a new compound, methyl 3beta,7beta,21beta-trihydroxyursa-9(11),12-dien-28-oate (22). Two other triterpenoids, 3beta,28-dihydroxyurs-12-ene (uvaol) (23) and 3beta,28-bis(dimethylcarbamoxy)urs-12-ene (24) were not transformed by the micro-organism, however.     

一个戊二烯酮化合物的合成及其杀菌抗癌活性

目的:合成戊二烯酮类化合物(1E,4E)-1,5-二(2-羟基苯基)-1 ,4-戊二烯-3-酮,并进行杀菌和抗肿瘤活性测定.方法:水杨醛和丙酮反 应得到目标产物;采用生长速率法和MTT比色法分别测定了该化合物的杀菌抗癌活性.结果:目标物结构经元素分析、红外光谱、核磁共振氢谱确证;该化合物在500 μg/mL浓度下对小麦赤霉病原菌、辣椒枯萎病原菌、苹果腐烂病原菌的抑制率分别为73.0% ±3.3%、70.4%±1.5%、79.2%±2.1%;在5.0ìmol/L下对PC-3细胞的72h的体外抑制率为71 .4%±2.7%.结论:该化合物有较好的杀菌抗癌活性,可以以它为先导进行结构优化,以期设计、合成出高效、选择性好的同类化合物.     

Inhibitors of sterol synthesis. Oleate ester of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one as a substrate for pancreatic cholesterol esterase

5 alpha-Cholest-8(14)-en-3 beta-yl-15-one oleate (15-ketosteryl oleate), the oleate ester of a compound with the capacity to lower serum cholesterol, was effectively hydrolyzed by partially purified porcine pancreatic cholesterol esterase with an apparent Km of 0.28 +/- 0.01 mM and a Vmax of 0.62 +/- 0.01 mumol/min per mg protein compared to an apparent Km of 0.19 +/- 0.02 mM and a Vmax of 0.37 +/- 0.02 mumol/min per mg protein for cholesteryl oleate. The 15-ketosteryl oleate was also hydrolyzed by highly purified rat pancreatic cholesterol esterase with an apparent Km of 0.20 +/- 0.01 mM and a Vmax of 86.7 +/- 3.0 mumol/min per mg protein compared to an apparent Km of 0.43 +/- 0.01 mM and a Vmax of 119.8 +/- 2.6 mumol/min per mg protein for cholesteryl oleate. 15-Ketosteryl oleate is, therefore, a good substrate for pancreatic cholesterol esterase from either source. The 15-ketosterol is a weak competitive inhibitor of partially purified porcine pancreatic cholesterol esterase when cholesteryl oleate is the substrate.     

Practical one-pot conversion of 17beta-estradiol to 10beta-hydroxy- (p-quinol) and 10beta-chloro-17beta-hydroxyestra-1,4-dien-3-one

An efficient one-pot procedure for the preparation of 10beta,17beta-dihydroxyestra-1,4-dien-3-one (p-quinol, 1, 75%) is reported, involving oxidation of 17beta-estradiol with potassium permanganate. Similar treatment of 17beta-estradiol with sodium chlorite led to 10beta-chloro-17beta-hydroxyestra-1,4-dien-3-one (2) in 44% yield along with smaller amounts 4-chloro-10beta,17beta-dihydroxyestra-1,4-dien-3-one (3), 2,10beta-dichloro-17beta-hydroxyestra-1,4-dien-3-one (4), and 4,10beta-dichloro-17beta-hydroxyestra-1,4-dien-3-one (5).