Androstenes, addition

Azide addition also occurs with the electron-poor olefinic double bond in androstene a mixture of the triazoline adduct and aziridine is obtained, the latter arising from the thermolysis of the triazoline formed by azide addition in the opposite direction (Scheme 74).251... 

The formation of the GSH conjugate (or the J4 isomer of androstene-3,17-dione) is monitored at 30°C in a jacketed spectrophotometer micro-cell. The GSH-containing sodium phosphate buffer (0.1 M, 0.9 ml) of appropriate GSH concentration, is added first to the cuvette followed by the substrate in 0.05 ml ethanol (or methanol for d5-an-drostene-3,17-dione). The reaction is initiated by the addition of the enzyme preparation in the appropriate sodium phosphate buffer and... 

This sequence was developed for conversion of 9a-hydroxy-4-androstene-3,17-dione, a biodegradation product of the common plant sterol -sitosteTol, to corticosteroids. The transposition of the 9a -hydroxy group to an 11 /3-hydroxy group can be accomplished readily by dehydration to the 9(ll)-ene, addition of HOBr, and reduction of the 9tr-bromine with chromium(II) chloride. 

The addition to steroidal alkenes promoted by chromium(II) chloride77 78, as well as thermal and photolytic additions, are predominantly trans 0. 2-Cholestene underwent thermal addition of ethyl dichlorocarbamate to give 2j8-chloro-3a-ethoxycarbonylaminocholestane80. Low yields of adducts were obtained from 3j8-acetoxy-5-androsten-17-one80. 

The metabolism of 17)3-hydroxy-l-methyl-A -androstene-3-one is markedly different from that of testosterone. In addition to the large amount of unchanged compound, small amounts of l-methyl-A -andro-stene-3,17-dione and la-methyl-androstane-3,17-dione also are excreted in the urine, but the 1-methylated androsterone or etiocholane derivatives are not found [275]. 

Metabolism studies. GC-MS is a powerful technique for following and identifying the metabolic products from the in vitro incubation of tissue preparations with steroid substrates. Examples of such studies include the 16a-hydroxylation of 18-hydroxydeoxycorticosterone by human adrenal gland [254], the eiromatization of 3jS,15, 16 -trihydroxy-5-androsten-17-one by placental homogenates [255], and the demonstration of 1/3, 12/3, 6a and 6/3 hydroxylase enzyme activities in microsomal preparations of human foetal hepatic tissue [256]. In the latter study, testosterone was used as substrate and in addition to the hydroxylated metabolites isolated, several other testosterone derivatives indicated the presence of 3a, 3/3 and 17/3-hydroxysteroid oxidoreductase in the adrenal gland preparation. 

The Palo Alto Syntex Research group8 effected addition of difluorocarbene to th double bond of 17/3-hydroxy-5a-A -androstene-3-one acetate (1) by drop wise addition of a saturated solution of 20-50 equivalents of sodium chlorodifluoroacetate in diglyme or triglyme to a 10% solution of the enone in the same solvent at 165-... 

The driving force behind the development of total syntheses for estrane and to some extent gonanes described in Chapters 2 and 3 lay in the then scarce and hence expensive steroid starting materials. The schemes that were developed made possible the elaboration of derivatives not accessible from estrone, such as gonanes with an additional carbon on the angular methyl at C13. Both androstene-17-dione and testosterone have been prepared by total synthesis. The schemes by which that was accomplished, however, were lengthy and complex. Those syntheses mainly represented a tour deforce for chemical synthesis since they were not competitive with sources of androstanes from pregnenolone or by fermentation. 

The 19-azido (93) and 19-methylthio (94) 4-androstene-3,17-diones have been found to be potent competitive reversible inhibitors (Kj = 5 nM and /fj = 1 nM respectively, for (androstenedione) = 25 nM). The same workers also discovered that 19-methanesulphonylthioandrostene-3,17-dione (95) inactivates AR in the presence of NADPH and Oj, although no kinetic or inhibitory data were presented [213]. The interaction of (94) with the active site was found to differ from that of (95) in that the latter displaces the substrate steroid from its binding site and on metabolism deactivates the enzyme, whereas (94) interacts with the substrate binding site but also with the haem-iron complex via a postulated coordinate bond. The differences in binding were deduced by examination of the ultraviolet spectral changes induced by the addition of the two inhibitors to the AR enzyme preparation. 

The distribution of 0 between the hydroxyl and hydroxymethylene functions of 19a and 19b, determined by the fragmentation pattern of these two species during mass spectrometry, could only result from addition of the carboxyl of the enzyme to the a face of the steroid. Had the carboxyl added to the spiro carbon from the jS face, the 0 would have been located in the hydroxymethylene function of 19a. Clearly, if the binding of the oxiranyl steroids is analogous to that of steroid substrates, Asp-38 could not be involved in the normal intramolecular proton transfer associated with the isomerization reaction. However, as recently emphasized by Pollack ei al., the oxiranyl steroids could conceivably bind to the active site in an upsidedown orientation in comparison to that of the substrate steroids (133). In this case, Asp-38 would still be the most probable base involved in the intramolecular proton transfer reaction. Perhaps upsidedown binding accounts for the report that the C-4a hydrogen of 5-androstene-3,17-dione undergoes slow labilization in the presence of the isomerase (134, 135). 

A soln. of Z i -androstene-3/ -ol-17-one acetate in the minimum amount of diloro-form distributed as a film on the inside walls of a large conical flask, which is then filled with oxygen, stirred 5 days at room temp, with addition of more chloroform to prevent crystallization of the startg. m. 14j5-hydroperoxy-zli5-androstene-3 -ol-17-one 3-monoacetate. Y 82%. A. Afonso, Can. J. Chem. 47, 3693 (1969). 

A soln. of 3,17-dioxo-zl -androsten-19-al in dry tetrahydrofuran added to a suspension of Mg-methoxide in liq. NHg, stirred 1 hr. at -33°, NH4CI added portion-wise, stirred an additional 10 min., and treated with methylene diloride followed by water zl5(i0)-estrene-3,17-dione. Y 75%. C. M. Siegmann and M. S. de Winter, R. 89, 442 (1970). 

Sites of Reaction. Reduction of the carbonyl groups at the 3- and 17-positions by yeasts (mainly Saccharomyces cerevisiae) were among the first microbial transformations recognized by Mamoli and Vercellone (M-549, M-551, V-1046). Estradiol was synthesized conveniently from estrone (W-1085) and testosterone from androstene-dione usii this method (M-543). FoUowii the early work, many additional examples of reduction at the 3- and 17-positions have been reported, and also instances of reaction at the 7-, 9-, 16-, 19-, 20-, 21-, and 22-positions. 

In addition to cholesterol, several other sterols bind to P450 7A1 and show some conversion to (imcharacterized) oxidation products, i.e., epi-cholesterols, 5-androstene-3y5-ol [1776]. 

Additional results were interpreted to indicate that the inactivation was A -3-ketosteroid dependent and was probably active site-directed. Cyclohexeneone which possesses the same chromphore as A -3-keto-steroids but is not a competitive inhibitor of the enzyme, stimulated photoinactivation only to a small extent even when present at more than ten times the concentration of compounds that markedly stimulated photoinactivation. It was noted that the first-order rate constant for photoinactivation increases with increasing affinity of the enzyme for the particular steroid, a pattern to be expected if the photoinactivation is active site-directed. In addition, the rate of photoinactivation promoted by 19-nortesto8terone acetate was decreased approximately 2-fold in the presence of 21 juM 3j8-hydroxy-5-androstene-17j8-carboxylic acid, a competitive inhibitor that does not support photoinactivation of the enzyme. This protective effect further strengthens the conclusion that photoinactivation is a catalytic site-directed reaction. 

A mixture of 17a-methyltestosterone and 10 equivalents of K-ferf-butoxide in ferf-butanol stirred 1.5 hrs. at room temp, under Ng, then quenched by rapid addition of 10%-acetic acid to the resulting slurry 17a-methyl-z1 -androsten-17yJ-ol-3-one. Y 65%. F. e. and limitations s. H. J. Ringold and S. K. Malhotra, Tetrah. Let. 1962, 669. 

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