Topical anti-androgenicity of a new 4-azasteroid in the hamster.

The topical anti-androgenic activity of L-651,580 (methyl 3-oxo-4-methyl-4-aza-5 alpha-androst-1-ene-17 beta-carboxylate) was established in a series of for experiments using castrated male hamsters. During each 21-day experiment, the animals received a daily subcutaneous injection of 40 micrograms testosterone propionate or 20 micrograms dihydrotestosterone propionate. Test compound in 25 microliters of gel was applied daily to the left flank organ. Compounds assayed included L-651,580, WIN 17,665 (17 alpha-propyltestosterone), and SH-434 (17 beta-hydroxy-1 alpha-methyl-17 alpha-propyl-5 alpha-androstan-3-one). Endpoints were flank organ area, sebaceous gland area, and prostate weight. Very similar results were obtained with L-651,580 and WIN 17,665. Daily doses of 0.25 mg or more of either compound usually produced a significant reduction in the areas of treated flank organs and sebaceous glands underlying treated flank organs. Neither compound caused significant changes in the area of the contralateral flank organs and sebaceous glands, which indicated they possess little or no systemic activity at topically effective treatment levels. In direct comparisons, SH-434 was less anti-androgenic than L-651,580 or WIN 17,665, although in one experiment, 0.5 mg/d of SH-434 significantly reduced the area of treated flank organs and sebaceous glands. Neither WIN 17,665 nor SH-434 caused a change in prostate weight; however, in one of four tests, a significant decrease was induced by the 0.5 mg/d level of L-651,580. The results of these experiments show that the topical anti-androgenicity of L-651,580 compares very favorably with that of WIN 17,665 and SH-434. They also indicate that the topical administration of effective dosage levels of L-651,580 causes few, if any, systemic effects.

Azasteroids: structure-activity relationships for inhibition of 5 alpha-reductase and of androgen receptor binding.

A series of steroids, primarily 4-azasteroids, were prepared and tested in vitro as inhibitors of human and rat prostatic 5 alpha-reductase and of binding of dihydrotestosterone to the rat androgen receptor. The primary structural modifications were changes of the A ring and of moieties attached at the C-17 position of the steroid nucleus. New A-ring modifications included the 4-cyano-3-oxo-delta 4 system in the carbocyclic series and 1 alpha-CN, 1 alpha-CH3, 1 alpha,2 alpha-CH2, 2 beta-F, 2-aza, 2-oxa, and A-homo changes in the 3-oxo-4-aza series. In addition, 4-azasteroids with a D-homo ring or methyl substitution at C-7 (alpha and beta) or C-16 (alpha and beta) were prepared. The majority of the C-17 substituents were prepared from reactive intermediates derived from the 17 beta-COOH. Enhanced 5 alpha-reductase inhibition in both the human and rat enzyme assays is seen with 4-CN substitution on 3-oxo-delta 4 steroids and with a C-17 side chain incorporating a lipophilically substituted semipolar group on the 4-aza-3-oxo-5 alpha-androstane nucleus. Fewer highly active compounds were found in the human enzyme assay than in the rat assay. Structural requirements for inhibition of the rat androgen receptor are much different from those for inhibition of the enzyme. The 17 beta-OH moiety enhances potency more than any other feature while introduction of double bonds at C-1 or C-5 in the azasteroid gives a small improvement. Azasteroids unsubstituted at the 4-position show greatly diminished receptor activity.

Prostatic effects induced in dogs by chronic or acute oral administration of 5 alpha-reductase inhibitors.

A series of 4-azasteroidal 5 alpha-reductase inhibitors was tested in dogs to determine the effect of chronic (35-44 day) oral administration on prostate size and histology and acute oral administration on prostatic concentrations of testosterone (T) and dihydrotestosterone (DHT). The extent to which the results of the two tests were correlated was also studied in order to see whether the acute test could be used to predict activity in the chronic test. Six delta 1 analogs of the potent 5 alpha-reductase inhibitor, 4-MA (17 beta-N,N-diethylcarbamoyl-4-aza-4-methyl-5 alpha-androstan-3-one) were uniformly active at low dosage levels (less than or equal to 3 mg/kg) in both types of assay whereas several C1-C2 saturated analogs exhibited little activity in the chronic test. The nature of the side chain and whether there was a methyl or a proton at 4-N did not dramatically influence the activity of delta 1 compounds. There was a broad general agreement between the results of the two kinds of test in that if a compound acutely decreased the prostatic concentration of DHT it was likely to reduce prostate size and alter prostatic histology when given on a chronic basis.

5 Alpha-reductase inhibitory and anti-androgenic activities of some 4-azasteroids in the rat.

Inhibition of 5 alpha-reductase and anti-androgenicity were studied in rats treated with various 4-azasteroids. The known inhibitor, N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) served as a reference compound, and analogs of this basic molecule were assayed. Enhancement of enzyme inhibitory potency was usually seen with delta 1 analogs, whereas reduction in activity was noted with substitutuents such as delta 5, a spirotetrahydrofuran ring at C-17 or 4-deaza groups. Many of the 4-azasteroids had a much greater oral anti-androgenic effect against testosterone propionate (TP) than dihydrotestosterone propionate (DHTP). This difference in activity versus the two androgens is believed to reflect the necessity for TP to undergo reduction to DHT before becoming capable of stimulating prostatic growth. Inhibition of 5 alpha-reductase by active compounds prevented the conversion, thereby producing an anti-androgenic effect. In this regard, certain delta 1 analogs of 4-MA, particularly those bearing a 17 beta-(N-tert butylcarbamoyl) group, proved very effective against TP but were relatively inactive versus DHTP.

Selenium dioxide oxidation of vitamin D3 acetate to a dimer of 1-oxotransvitamin D3 acetate.

The treatment of vitamin D3 acetate with selenium dioxide and t-butyl hydroperoxide leads to a mixture from which a Diels-Alder dimer of 1-oxotransvitamin D3 acetate was isolated.

Species differences in prostatic steroid 5 alpha-reductases of rat, dog, and human.

The conversion of testosterone to 5 alpha-dihydrotestosterone by prostate particulates from rats, dogs, and humans was investigated, and significant species differences were found with their pH profiles, affinities for 4-azasteroidal inhibitors, and sensitivities to mercuric sulfhydryl reagents. The pH optima for the rat (pH 7), the dog (pH 6), and the human (pH 5) enzyme are significantly different. Mersalyl acid and p-hydroxymercuribenzoate inactivate only the rat 5 alpha-reductase, but not the human or dog enzyme. The rank orders of potencies of 24 3-oxo-4-azasteroids to inhibit 5 alpha-reductases of the rat, dog, and human prostate are different. The variation of the 17 beta-functional groups of the inhibitors demonstrates clearly the species differences. Those inhibitors with a 17 beta-diethylcarbamoyl, 17 beta-diisopropylcarbamoyl, 17 beta-t-butylcarbamoyl, or 17 beta-secbutylcarbonyl functional group are approximately equipotent as inhibitors of the rat and human enzymes, whereas they are only 0.1-15% as potent as inhibitors of the dog enzyme. On the other hand, those inhibitors with a 17 beta-spiroether functional group are most potent as inhibitors of the rat enzyme, are 15-50% as potent as inhibitors of the dog enzyme, and are 0.2-0.4% as potent as inhibitors of the human enzyme. Those inhibitors with a 17 beta-n-octylcarbamoyl, 17 beta-(1-carboxyethyl), or 17 beta-(1-carboxy-3-butyl) functional group are 2-3 orders of magnitude less potent as inhibitors of the dog and human enzymes than as inhibitors of the rat enzyme. These results suggest that prostatic 5 alpha-reductases of rats, dogs, and humans are significantly different. In spite of the significant species differences in inhibitor affinities, where determined, inhibition of the rat, dog and human enzymes by these compounds is competitive with testosterone. These 3-oxo-4-azasteroids have a similar rank order of potency as inhibitors of 5 alpha-reductase in human normal, benign hyperplastic, and cancerous prostates, indicating that the inhibitor-binding sites of 5 alpha-reductase in the prostate in different pathological states are similar. The affinities of the 3-oxo-4-azasteroids for rat prostatic cytosol receptor were determined. Five of these 5 alpha-reductase inhibitors have no significant affinity for the androgen receptor, whereas others do have an affinity for the receptor.

Synthesis and antiherpetic activity of (S)-, (R)-, and (+/-)-9-[(2,3-dihydroxy-1-propoxy)methyl]guanine, linear isomers of 2'-nor-2'-deoxyguanosine.

Racemic 9-[(2,3-dihydroxy-1-propoxy)methyl]guanine [(+/-)-iNDG], a new analogue of acyclovir (ACV) and a structural analogue of 2'-nor-2'-deoxyguanosine (2'NDG), was synthesized and found to inhibit the replication of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Subsequently, its optical isomers, (R)- and (S)-iNDG, were prepared from chiral intermediates. The chloromethyl ethers of 1,2-di-O-benzyl-D- and -L-glycerol were made and reacted with tris(trimethylsilyl)guanine to give the 9-alkylated guanines, which were deprotected by catalytic hydrogenolysis. Against HSV-1 and HSV-2 in cell culture, (S)-iNDG was approximately 10- to 25-fold more active than the R enantiomer and had an ED50 comparable to those for ACV and 2'NDG. The inferior activity of (R)-iNDG paralleled the poor inhibition of viral DNA polymerase by its phosphorylation products. In mice infected intraperitoneally or orofacially with HSV-1 or intravaginally with HSV-2, (S)-9-[(2,3-dihydroxy-1-propoxy)methyl]guanine [(S)-iNDG] was less efficacious than 2'NDG but comparable to or more active than ACV.

Photoaffinity labeling of steroid 5 alpha-reductase of rat liver and prostate microsomes.

21-Diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione (Diazo-MAPD) inhibits steroid 5 alpha-reductase in liver microsomes of female rats with a Ki value of 8.7 +/- 1.7 nM, and the inhibition is competitive with testosterone. It also inhibits the binding of a 5 alpha-reductase inhibitor, [3H] 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA), to the enzyme in liver microsomes. The inhibition of 5 alpha-reductase activity and of inhibitor binding activity by diazo-MAPD becomes irreversible upon UV irradiation. [1,2-3H]Diazo-MAPD binds to a single high affinity site (Kd 8 nM, 125 pmol binding sites/mg of protein) in liver microsomes of female rats, and this binding requires NADPH. Without UV irradiation, this binding is reversible, and it becomes irreversible upon UV irradiation. Both the initial reversible binding and the subsequent irreversible conjugation after UV irradiation are inhibited by inhibitors (diazo-MAPD and 4-MA) and substrates (progesterone and testosterone) of 5 alpha-reductase, but they are not inhibited by 5 alpha-reduced steroids (5 alpha-dihydrotestosterone and 5 alpha-androstan-3 alpha, 17 beta-diol). NADPH stimulates the binding of [3H] diazo-MAPD to microsomes of male rat liver and prostate. UV irradiation also induces conjugation of [3H] diazo-MAPD to these microsomes. Photoaffinity labeled liver microsomes of female rats were solubilized and fractionated by high performance gel filtration. The radioactive conjugate eluted in one major peak at Mr 50,000.

Azasteroids as inhibitors of rat prostatic 5 alpha-reductase.

A series of A-ring heterocyclic steroids has been prepared and tested for inhibition of rat prostatic steroid 5 alpha-reductase in vitro. Strikingly high inhibitory activity was found with a group of 17 beta-substituted 4-methyl-4-aza-5 alpha-androstan-3-ones. These compounds were prepared from 3-keto-delta 4-precursors by oxidative (O3 or NaIO4-KMnO4) A-ring cleavage followed, in turn, by ring closure with an amine and hydrogenation over platinum catalyst. Other A-ring azasteroids were made by Beckmann rearrangement of oximes of 2-oxo-A-nor, 3-oxo- and 4-oxo-5 alpha-androstanes. An A-nor-2-oxo-3-azasteroid was prepared by oxidative decarbonylation of a precursor 2,3-dioxo-4-azasteroid with m-chloroperbenzoic acid. A-ring modifications of the 4-azasteroids included delta 1-unsaturation, 2- and 4-substituents, and 3-carbonyl replacements. Side chains at the 17-position were varied with an emphasis on carboxylate derivatives (salts, esters, and amides).

4-Azasteroidal 5 alpha-reductase inhibitors without affinity for the androgen receptor.

In efforts to develop potent 5 alpha-reductase inhibitors without affinity for the androgen receptor, synthetic 3-oxo-5 alpha-steroids were tested for their ability to inhibit 5 alpha-reductase, using [14C]testosterone as the substrate, and for their ability to inhibit the binding of [3H]5 alpha-dihydrotestosterone to the androgen receptor of rat prostate cytosol. 2',3' alpha-Tetrahydrofuran-2'-spiro-17-(5 alpha-androstan-3-one) is not an inhibitor of 5 alpha-reductase and has a high affinity for the androgen receptor; substitution of the -CH2- at the 4-position with N-H resulted in a good inhibitor of 5 alpha-reductase. The 4-N-CH3 derivative is even more active, whereas the N-CH2-CH3 derivative is inactive. These 4-aza derivatives have much lower affinity for the androgen receptor than the parent compound. The 4-N-H derivatives of several 3-oxo-5 alpha-steroids were found to be 20-100% as potent as their corresponding 4-N-CH3 analogs as inhibitors of 5 alpha-reductase, whereas their androgen receptor affinities were at least 40-fold lower than their 4-N-CH3 analogs. Their 5 beta-isomers did not inhibit either 5 alpha-reductase or the androgen receptor binding of [3H]5 alpha-dihydrotestosterone. Two of these 4-N-H steroids, 17 beta-N,N-diethylcarbamoyl-4-aza-5 alpha-androstan-3-one and 17 beta-N, N-diisopropylcarbamoyl-4-aza-5 alpha-androstan-3-one, are potent 5 alpha-reductase inhibitors with Ki values equal to 29.2 +/- 1.7 and 12.6 +/- 0.8 nM, respectively, but have little affinity for the androgen receptor. The inhibition of 5 alpha-reductase by both compounds is competitive with testosterone. When [3H]testosterone was incubated with minced rat prostate in the presence of either of these two 4-azasteroids, the nuclear concentration of 5 alpha-dihydrotestosterone decreased and that of testosterone increased. The total nuclear uptake of testosterone plus 5 alpha-dihydrotestosterone was not significantly affected. These 4-azasteroids should be useful for investigating the importance of 5 alpha-reductase in androgen action in vivo.

Effect of a new 5 alpha-reductase inhibitor on size, histologic characteristics, and androgen concentrations of the canine prostate.

Mature male beagles were used in studies designed to determine the effect of the steroidal 5 alpha-reductase inhibitor 17 beta-N,N-diethylcarbamoyl-4-aza-4-methyl-5 alpha-androstan-3-one (4-MA) on size, histology, and androgen concentration of the prostate. Subcutaneous administration of 3 or 15 mg/kg/day for 43 days caused a sharp decline in prostate volume. flattening of prostatic epithelial cells, vacuolization of the cytoplasm and pycnosis of the nuclei. Whereas serum testosterone levels remained normal in dogs injected with 3 mg/kg/day, they were lower in those that received 15 mg/kg/day. Concentration of both testosterone and 5 alpha-dihydrotestosterone were reduced in the prostates of dogs that had received either 3 or 15 mg/kg/day of 4-MA. The 15 mg/kg/day level also appeared to adversely affect spermatogenesis. In a 43-day study, 4-MA given orally once each day at levels of 0.1, 0.3, or 1 mg/kg failed to cause a significant decrease in prostate volume. However, daily divided oral doses totaling 1 or 3 mg/kg were given in a 42-day test and both treatment levels produced significant reductions in prostate size.

Antiandrogens. 2',3' -Tetrahydrofuran-2'spiro-17-(1,2 -methylene-4-androsten-3-ones).

PIP: The syntheses of several 1,2alpha-methylene steroids containing a sp irotetrahydrofuran ring at the 17 position are described. The antiandro genic activity of these compounds was determined in immature male castrate rats treated with testosterone enanthate. The ability of the compounds to antagonize the androgen-stimulated weight gain of the simin al vesicle and ventral prostate serves as a measure of their activity. From the results, the antiandrogenic activity associated with the Tetrah ydrofuran-2'-spiro-17 androstenones I and II has been increased by addit ion of a 1,2alpha-methylene function and by incorporation of either a 6- chloro-delta6 or 6,7alpha-difluoromethylene group to the basic steroid s keleton. These compounds show minimal other hormonal activity. Tert-Butyl chromate oxidation of the spirotetrahydrofuran XIII affords the corresponding spirolactone XIV in high yield.^ieng