New Aromatic Esters of Progesterone as Antiandrogens

The in vivo and in vitro antiandrogenic activity of four new progesterone derivatives: 4-bromo-17α-(p-fluorobenzoyloxy)-4-pregnene-3,20-dione 1,4-bromo-17α-(p-chlorobenzoyloxy)-4-pregnene-3,20-dione 2, 4-bromo-17α-(p-bromobenzoyloxy)-4-pregnene-3,20-dione 3 and 4-bromo-17α-(p-toluoyloxy)-4-pregnene-3, 20-dione 4 was determined. These compounds were evaluated as antiandrogens on gonadectomized hamster prostate and reduced the weight of the prostate glands in gonadectomized hamsters treated with testosterone 5 (T) or dihydrotestosterone 6 (DHT) in a similar manner to that of commercially available finasteride, thus indicating a potent in vivo effect. The in vitro studies showed that steroids 1–4 have a weak inhibitory activity on 5α-reductase with IC50 values of: 280 (1), 2.6 (2), 1.6 (3) and 114?μM (4). The presence of Cl and Br atoms in the C-17 benzoyloxy group tends to increase the inhibitory potency of the compounds.

The binding efficiency of the synthesized steroids 1–4 to the androgen receptor of the prostate gland is also evaluated. All compounds form a complex with the receptor and this explains the weight reduction of the seminal vesicles in the animals treated with DHT plus steroids 1–4.     

Relative binding affinity of novel steroids to androgen receptors in hamster prostate

The in vivo and in vitro antiandrogenic activity of four aromatic esters 10a-10d, one aliphatic ester 10e based on the pregna-4,16-diene-6, 20-dione structure and two aromatic 17c, 17d and two aliphatic valeroyloxy esters 17a, 17b based on the more saturated 4-pregnene-6,20-dione skeleton was examined. The biological activity of steroids 9, 10a-10e and 17a-17d, was determined using prostate glands from gonadectomized adult male golden hamsters. In the in vitro studies, the relative binding affinity of these steroids to cytoplasmic androgen receptor (AR) of hamster prostate was determined from, the corresponding IC50 values obtained from the competitive binding plots. The standards dihydrotestosterone (DHT) and cyproterone (CA) acetate used have displaced [3H]DHT from the AR with an IC50 value of 3.2 and 4.4 nM respectively. All steroidal compounds synthesized in this study showed a binding affinity for the androgen receptor, present in the cytosol from prostate hamster; compounds 10a-10c showed the highest affinities for this receptor. The in vivo experiments showed that all steroidal derivatives were subcutaneously active, since they decreased the weight of the prostate gland in gonadectomized hamsters treated with DHT, and are antagonists for the androgen receptor since they block the DHT-induced prostate weight gain. The derivatives having the more conjugated 4,16-pregnadiene-6, 20-dione system (10a-10c) exhibited a higher antiandrogenic activity than the corresponding steroids (17a-17d) based on the more saturated 4-pregnene-6,20-dione system.     

Steroidal 5α-reductase inhibitors using 4-androstenedione as substrate

The aim of this study was to determine the capacity of some progesterone derivatives, to inhibit the conversion of labeled androstenedione ([(3)H] 4-dione) to [(3)H]dihydrotestosterone ([(3)H]DHT) in prostate nuclear membrane fractions, where the 5α-reductase activity is present. The enzyme 5α-reductase catalyzes the 5α-reduction of 4-dione whereas the 17β-hydroxysteroid dehydrogenase catalyzes the transformation of 4-dione to testosterone or 5α-dione to dihydrotestosterone (DHT). Moreover, we also investigated the role of unlabeled 5α-dione in these pathways. In order to determine the inhibitory effect of different concentrations of the progesterone derivatives in the conversion of [(3)H] 4-dione to [(3)H]DHT, homogenates of human prostate were incubated with [(3)H] 4-dione, NADPH and increasing concentrations of non-labeled 5α-dione. The incubating mixture was extracted and purified using thin layer chromatography. The fraction of the chromatogram corresponding to the standard of DHT was separated and the radioactivity determined. The results showed that the presence of [(3)H] 4-dione plus unlabelled 5α-dione produced similar levels of DHT as compared to [(3)H] 4-dione. On the other hand, the results indicated that 17α-hydroxypregn-4-ene-3,20-dione 5 and 4-bromo-17α-hydroxypregn-4-ene-3,20-dione 7b, were the most potent steroids to inhibit the conversion of [(3)H] 4-dione to [(3)H]DHT, showing IC(50) values of 2 and 1.6?nM, respectively.     

Molecular interactions of progesterone derivatives with 5α-reductase types 1 and 2 and androgen receptors

The aim of this study was to ascertain the inhibitory effect of several progesterone derivatives for 5α-reductase types 1 and 2 isozymes and to determine the binding to the androgen receptor.The 3,20-dioxopregna-4-ene-17α-yl acetate 4 containing an acetoxy group in C-17 and steroid 17α-hydroxypregn-4-ene-3,20-dione 5 having a hydroxyl group in the same position inhibited both isozymes. On the other hand, 17α-hydroxy-4,5-epoxypregnan-3,20-dione 6 with an epoxy function at C-4, inhibited only the type 1 enzyme. Steroid 4-chloro-17α-hydroxypregn-4-ene-3,20-dione 7a and 4-bromo-17α-hydroxypregn-4-ene-3,20-dione 7b having the C-4 conjugated system and a chlorine or a bromine atom at C-4 respectively, inhibited both types of 5α-reductase. These results indicate that an increase in the electronegativity of ring A produces a major inhibitory activity for 5α-reductase type 1; however this increase was not observed for type 2 enzyme. When the free hydroxyl group of 7a or 7b was esterified, compounds 3,20-dioxo-4-chloropregn-4-ene-17α yl-4-ethylbenzoate 8a and 3,20-dioxo-4-bromopregn-4-ene-17α yl-4-ethylbenzoate 8b were obtained; these steroids inhibited only the 5α-reductase type 2 enzyme.Finasteride and steroids 4, 5, 7b, 8a showed a comparable in vivo pharmacological activity, however the IC₅₀ values of these compounds were higher as compared to that of finasteride.These results indicated also that steroids 4, 5, 7a, and 7b bind to the androgen receptor whereas compounds 6, 8a and 8b failed to do so.The overall data from this study showed that steroids 5 and 7b bind to the AR and decreased of the growth of prostate and seminal vesicles. Moreover, 4 decreased also the growth of seminal vesicles.     

Androgenic and anti-androgenic effects of progesterone derivatives with different halogens as substituents at the C-6 position.

The pharmacological activities of four pregnane derivatives: 17alpha-hydroxy-16beta-methylpregna-4,6-diene-3,20-dio ne (7), 17alpha-acetoxy-16beta-methylpregna-4,6-diene-3,20-dio ne (8), 17alpha-acetoxy-6-bromo-16beta-methylpregna-4,6-diene- 3,20-dione (10), and 17alpha-acetoxy-6-chloro-16beta-methylpregna-4,6-diene -3,20-dione (11), were determined. The derivatives were evaluated on gonadectomized male hamster flank organs and seminal vesicles. The results indicate that topical applications of testosterone (T) on the flank organs increased the diameter of the pigmented spot. Similarly, the same phenomenon occurred on the glands treated with compound 11, whereas compound 10 decreased the size of the spot significantly. In this study, we determined the effects of several new steroids on the conversion of T to DHT in flank organs and seminal vesicles. The results show that compound 10 inhibited T conversion to DHT, but compound 11, at a dose of 200 microg, stimulated T conversion in both flank organs and seminal vesicles. However, when 2 mg of compound 11 was applied, it inhibited the conversion of T to DHT, suggesting that this compound also represses gonadotropin release. The difference between compounds 10 and 11 involves the electronegativity of the halogen at the C-6 position of the progesterone skeleton. These data clearly indicate that by decreasing the electronegativity of the halogen at C-6 (compound 10), 5alpha-reductase is inhibited in both tissues and at different pHs. On the other hand, when the electronegativity of the halogen atom was increased (11), there was a much lower inhibitory effect on the conversion of T to DHT.     

Steroidal 5α-reductase inhibitors using 4-androstenedione as substrate

The aim of this study was to determine the capacity of some progesterone derivatives, to inhibit the conversion of labeled androstenedione ([³H] 4-dione) to [³H]dihydrotestosterone ([³H]DHT) in prostate nuclear membrane fractions, where the 5α-reductase activity is present. The enzyme 5α-reductase catalyzes the 5α-reduction of 4-dione whereas the 17β-hydroxysteroid dehydrogenase catalyzes the transformation of 4-dione to testosterone or 5α-dione to dihydrotestosterone (DHT). Moreover, we also investigated the role of unlabeled 5α-dione in these pathways. In order to determine the inhibitory effect of different concentrations of the progesterone derivatives in the conversion of [³H] 4-dione to [³H]DHT, homogenates of human prostate were incubated with [³H] 4-dione, NADPH and increasing concentrations of non-labeled 5α-dione. The incubating mixture was extracted and purified using thin layer chromatography. The fraction of the chromatogram corresponding to the standard of DHT was separated and the radioactivity determined. The results showed that the presence of [³H] 4-dione plus unlabelled 5α-dione produced similar levels of DHT as compared to [³H] 4-dione. On the other hand, the results indicated that 17α-hydroxypregn-4-ene-3,20-dione 5 and 4-bromo-17α-hydroxypregn-4-ene-3,20-dione 7b, were the most potent steroids to inhibit the conversion of [³H] 4-dione to [³H]DHT, showing IC₅₀ values of 2 and 1.6?nM, respectively.     

Identification of 2 alpha-hydroxy-4-pregnene-3,20-dione in human pregnancy urine.

2 alpha-Hydroxyprogesterone (2 alpha-hydroxy-4-pregnene-3,20-dione) was identified in human late pregnancy urine by liquid-gel chromatography, GLC and GC-MS. In addition, the following 2-hydroxylated C21 steroids were found and identified as 2 zeta-hydroxy-5 zeta-pregnane-3,20-dione, 2 zeta,20 zeta-dihydroxy-4-pregnen-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha- (and 5 beta)-pregnan-20-one, two isomers of pregnane-2,3,20-triol and 2 zeta,3 zeta,16 zeta-trihydroxy-5 zeta-pregnan-20-one.     

In vivo and in vitro effect of novel 4,16-pregnadiene-6,20-dione derivatives, as 5alpha-reductase inhibitors

In this study, we report the synthesis and biological evaluation of several new 3-substituted pregna-4,16-diene-6,20-dione derivatives (11a-11d). These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological effect of these steroids was demonstrated in in vivo and in vitro experiments. In the in vivo experiments, we measured the activity of the 11a-11d on the weight of the prostate gland of gonadectomized hamsters treated with testosterone plus finasteride or with the new steroids. For the studies in vitro, we determined the IC50 values by measuring the steroid concentration that inhibits 50% of the activity of 5alpha-reductase present in human prostate. In order to study the mechanism of action of 11a-11d, we also determined the capacity of these steroids to bind to the androgen receptor (AR) present in the rat prostate cytosol using labeled mibolerone as a tracer. The results from this work indicated that compounds 11a-11d significantly decreased the weight of the prostate as compared to testosterone treated animals and this reduction of the weight of the prostate was comparable to that produced by the finasteride. On the other hand 11a-11d exhibited a high inhibitory activity for the human 5alpha-reductase enzyme with IC50 values of 1.4 x 10(-8), 1.8 x 10(-9), 1.0 x 10(-8) and 4 x 10(-5) respectively. However the IC50 value of 11a (1.8 x 10(-9)) was the only one lower than that of finasteride (8.5 x 10(-9)). Nevertheless this compound did not show a higher potency in vivo as compared to that of compounds 11b-11d. The competition analysis for the androgen receptor indicated that the IC50 value of non-labeled mibolerone used in this experiment was 1nM, whereas steroids 10, 11a-11d did not inhibit the labeled mibolerone binding to the androgen receptor. On the other hand, steroid 10 did not show any activities in vitro or in vivo, and for this reason these steroidal derivatives (11a-11d) cannot be considered as prodrugs of compound 10. In conclusion, the compounds containing chlorine 11a, bromine 11b, iodine 11c atoms, and 11d (without any substituent in the ester moiety) at C-3 produce a significant decrease of the prostate weight in castrated animals treated with T and inhibits the activity of the 5alpha-reductase. Apparently the presence of the halogen atoms in compounds 11a-11c enhances the inhibitory activity for the 5alpha-reductase enzyme.     

Steroid transformations by species of Cephalosporium and other fungi

A total of 58 cultures, tentatively identified as species of the genus Cephalosporium, were screened in flask fermentations for their ability to effect conversions of progesterone (Delta(4)-pregnene-3,20-dione) and Reichstein's Substance S (Delta(4)-pregnene-17alpha,21-diol-3,20-dione). A large number of transformations were observed by means of a series of five paper chromatography systems rated for analysis of steroid compounds ranging in polarity from progesterone to polyhydroxylated steroids. Five different transformation products were selected for isolation and identification. For purposes of recovery, conversions were conducted under submerged conditions in either 4- or 200-liter fermentors in which the broth was agitated and aerated. The steroid substrate was dissolved in acetone and added aseptically to the growing culture in a final concentration of 0.025%. After the conversions were effected, the whole broth was extracted with chloroform, and the transformation products were recovered, either by direct crystallization from solvents or through the use of silica gel columns. It was determined that C. ciferrii 21C converted progesterone to Delta(4)-androstene-3,17-dione. Kendall's Compound F (Delta(4)-pregnene-11beta,17alpha,21-triol-3,20-dione) was converted to its 20beta-ol analogue by Geotrichum sp. 51C (during these studies, a number of cultures were taxonomically reclassified). Cephalosporium sp. 27C formed the Delta(1)-analogue of Reichstein's Substance S, and Cephalosporium sclerotigenum 31C and Verticillium aphidum both converted Substance S to the 6beta-hydroxy derivative. Paecilomyces persicinus 22C converted Substance S to a product believed to be a dihydroxylated derivative.     

Synthesis and characteristics of allylic 4-pregnene-3,20-diols found in gonadal and breast tissues

Recently several allylic steroids have been found in gonadal and breast tissues. In order to establish their presence and identity in tissues and determine the possible biological properties, a method for the synthesis of 4-pregnene-3 alpha,20 alpha-diol, 4-pregnene-3 alpha, 20 beta-diol, 4-pregnene-3 beta,20 alpha-diol, and 4-pregnene-3 beta,20 beta-diol was developed using 4-pregnene-3,20-dione (progesterone) as substrate and freshly-prepared aluminum isopropoxide in isopropyl alcohol as reducing agent. The yields were about 19%, 30%, 13%, and 38% for the 3 alpha,20 alpha-, 3 alpha,20 beta-, 3 beta,20 alpha-, and 3 beta,20 beta-diols, respectively. The structures and stereochemistry of these diols were established using proton and carbon NMR spectroscopy and infrared and mass spectrometry.     

Metabolism of 11-deoxycortisol by a Bacillus species

Microbial transformation by a Bacillus species was employed for the preparation of potentially important derivatives of 11-deoxycortisol. Each microbial metabolite was characterised by the application of various spectroscopic methods. The five metabolites of 11-deoxycortisol were characterised as 4-androstene-3,17-dione (2), 14-hydroxy-4-androstene-3,17-dione (3), 14,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (4), 6 beta,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (5) and 15 alpha,17 alpha,21-trihydroxy-4-pregnene-3,20-dione (6). The availability of the metabolites enabled complete elucidation of their [13C]NMR spectra.     

6-Oxygenation of 3-oxo-4-ene-steroids in high yields after 3-imine-formation

3-Imine formation between primary amines and 3-oxo-4-ene-steroids, followed by hydrolysis of the imines (either spontaneously during work up or induced by acetic acid) has been shown to cause 6-oxygenation of the steroids tested (17 beta-hydroxy-4-androsten-3-one, 4-androstene-3,17-dione, 4-pregnene-3,20-dione and 4-cholesten-3-one). The main products are the 6 beta-hydroxy- and the 6-oxo-derivatives of the respective steroid. These derivatives were identified by chromatographic mobilities and by gas chromatography-mass spectrometry. The formation of 6 beta-hydroperoxy-derivatives is suggested and these derivatives were tentatively identified. The highest yields of 6-oxygenated products (30-50%) were found when cadaverine and spermine were reacted with the steroids. The addition of reduced glutathione during hydrolysis of the steroid 3-imines of cadaverine, hexylamine and ethanolamine as well as addition of ascorbic acid during the hydrolysis of the steroid 3-imines of cadaverine substantially reduced the 6-oxygenation. Steroid 3-imine formation and hydrolysis which yields 6-oxygenated derivatives has also been shown to occur during work up (evaporation) of organic solvent extracts of rat liver microsomes (105,000 g sediments) to which 17 beta-hydroxy-4-androsten-3-one, 4-androstene-3,17-dione, 4-pregnene-3,20-dione or 4-cholesten-3-one respectively had been added. It is concluded that there is a risk that these organic reactions are mistaken for enzymatic conversions during in vitro investigations of 3-oxo-4-ene-steroids.     

Formation of 5alpha-reduced C19 steroids from progesterone in vivo by 5alpha-reduced pathway in older prepubertal rat testis.

Either [3H] progesterone (0.5 or 5 nmol/5 muCi), 5alpha-[3H] pregnane-3,20-dione (5 nmol/5 muCi) or [14C] progesterone (6.6 nmol/0.2 muCi) plus 5alpha-[3H]-pregnane-3,20-dione (1 or 6.6 nmol/0.6 muCi), suspended in 0.05 ml of physiological saline solution, was injected into each testis of 32- and 90-day-old rats. Following injection, radioactive metabolites in testis and spermatic vein blood were extracted, isolated, measured and identified by column and paper chromatographies, with derivative formation and recrystallization to constant specific activity. In the blood and testis of older prepubertal rats, major 17-OH-C21 and C19 metabolites of progesterone were 5alpha-reduced steroids such as 3alpha, 17alpha-dihydroxy-5alpha-pregnan-20-one, 5alpha-androstane-3alpha,17beta-diol and androsterone. Following injection of [14C] progesterone plus 5alpha-[3H] pregnane-3,20-dione into 32-day-old rat testis, no significant augmentation of the isotope from progesterone was observed in 5alpha-reduced C19 steroids as compared with 5alpha-reduced 17-OH-C21 steroids, indicating that 5alpha-reduced C19 steroids were mainly formed from 5alpha-reduced 17-OH-C21 steroids in older prepubertal testis. In the blood and testis of adult rats, small amounts of 5alpha-reduced metabolites were shown to be produced from progesterone, while active 17alpha-hydroxylation of 5alpha-pregnane-3,20-dione followed by C17-C20-lyase reaction was demonstrated. These findings seem to indicate that formation of 5alpha-reduced C19 steroids from progesterone by the 5alpha-reduced pathway is a major pathway of androgen biosynthesis in older prepubertal rat testis in vivo.     

New 5alpha-reductase inhibitors: in vitro and in vivo effects

The enzyme 5alpha-reductase is responsible for the conversion of testosterone (T) to its more potent androgen dihydrotestosterone (DHT). This steroid had been implicated in androgen-dependent diseases such as: benign prostatic hyperplasia, prostate cancer, acne and androgenic alopecia. The inhibition of 5alpha-reductase enzyme offers a potentially useful treatment for these diseases. In this study, we report the synthesis and pharmacological evaluation of several new 3-substituted pregna-4, 16-diene-6, 20-dione derivatives. These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological activity of the new steroidal derivatives was determined in vivo as well as in vitro experiments. In vivo experiments, the anti-androgenic effect of the steroids was demonstrated by the decrease of the weight of the prostate gland of gonadectomized hamster treated with T plus finasteride or the new steroids. The IC50 value of these steroids was determined by measuring the conversion of radio labeled T to DHT. The results of this study carried out with 5alpha-reductase enzyme from hamster and human prostate showed that four of the six steroidal derivatives (5, 7, 9, 10) exhibited much higher 5alpha-reductase inhibitory activity, as indicated by the IC50 values than the presently used Proscar 3 (finasteride). The comparison of the weight of the hamster's prostate gland indicated that compound 5 had a comparable weight decrease as finasteride. The overall data of this study showed very clearly those compounds 5, 7, 9, 10 are good inhibitors for the 5alpha-reductase enzyme.     

New progesterone derivatives as inhibitors of 5alpha-reductase enzyme and prostate cancer cell growth

In this study we report the synthesis and pharmacological evaluation, in vivo as well as in vitro, of four new progesterone derivatives 4-7. The evaluation in vivo was carried out on gonadectomized male hamsters that were injected subcutaneously daily with 1 mg/Kg of testosterone (T) and/or 1 mg/Kg of finasteride, or with 2 mg/Kg of the novel compounds. It was observed that when testosterone (T) and finasteride or compound 4 were injected together, the weight of the prostate decreased significantly as compared to that oftestosterone-treated animals. Compounds 5-7 did not show any in vivo activity. The 5alpha-reductase inhibitory activity of the novel compounds was determined in vitro using human prostate homogenates; the steroids 4-7 inhibited the 5alpha-reductase activity with IC50 values lower than that for the reference compound finasteride. 3. The effect of compounds 4-7 on the growth of lymphocytes and prostate cancer culture cells line was that steroid 4 inhibited the growth of both cells lines at a concentration of 50 microM and showed a cytotoxic effect whereas compounds 5-7 showed a much lower inhibition. Nevertheless steroids 4-7 didn't exhibit any toxic effects in vivo since the animals remained alive during the six days of treatment.     

Effect of 5 alpha-dihydrocorticoids on enzymes of gluconeogenesis in rat liver

5 alpha-Dihydrocortisol (11 beta, 17, 21-trihydroxy-5 alpha-pregnane-3,20-dione), 5 alpha-dihydrocorticosterone (11 beta, 21-dihydroxy-5 alpha-pregnane-3,20-dione) as well as cortisol (11 beta, 17, 21-trihydroxy-4-pregnene-3,20-dione) and corticosterone (11 beta, 21-dihydroxy-4-pregnene-3,20-dione) were administered for seven days to male rats. Blood glucose increased in cortisol- and corticosterone-treated rats and blood insulin decreased after 5 alpha-dihydrocorticosteroid treatment. In the liver, total protein was elevated after cortisol, corticosterone and 5 alpha-dihydrocorticosterone application. Phosphoenolpyruvate carboxykinase and fructose-1,6-diphosphatase activities in liver were significantly lowered after treatment with 5 alpha-dihydrocortisol and 5 alpha-dihydrocorticosterone.     

Various 18-substituted progesterones

In order to test the potential biological activity of 18-substituted progesterones, 3,20-dioxo-4-pregene-18-carbonitrile (ld approximately) was converted to 3,20-dioxo-4-pregnene-18-carboxylic acid (lb approximately) and methyl 3,20-dioxo-4-pregnene-18-carboxylate (ld approximately) via a sequence of reactions involving an intramolecular hydrolysis of the 18--arbonitrile. Lithium aluminum hydride reduction of the bisethylene ketal derived from la approximately furnished 18-aminomethyl-5-pregnene-3,20-dione 3,20-bisethylene ketal (8 approximately). Acetylation and hydrolysis furnished 18-acetamidomethyl-4-pregnene-3,20-dione (lf approximately) and simple hydrolysis of 8 approximately furnished 3'alpha H-5' 6'-dihydro-2',19 beta-dimethyl-3-oxo-4-goneno [13,17-c]pyridine (9 approximately). None of the compounds exhibited any activity in Clauberg or anti-Clauberg tests.     

Synthesis of the allylic gonadal steroids, 3 alpha-hydroxy-4-pregnen-20-one and 3 alpha-hydroxy-4-androsten-17-one, and of 3 alpha-hydroxy-5 alpha-pregnan-20-one

A method for the convenient synthesis of the recently isolated allylic gonadal steroids, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha-dihydroprogesterone; 3 alpha-DHP) and 3 alpha-hydroxy-4-androsten-17-one (3 alpha-HA), was developed using 4-pregnene-3,20-dione (progesterone) and 4-androstene-3,17-dione as substrates and potassium trisiamylborohydride (KS-Selectride) as reducing agent. Similar reactions were also used for the reduction of 5 alpha-pregnane-3,20-dione to 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-HP). The yields were about 15%, 50%, and greater than 90% for 3 alpha-DHP, 3 alpha-HA and 3 alpha-HP, respectively. Structures of the products, including the 3 beta-isomers and the 17 alpha-epimer, formed in these reactions were determined by NMR and mass spectroscopic methods.     

Prostate. III--A structural feature characteristic of the rat prostate 5 alpha-reductase active site

To aid in the design of new inhibitors of steroidal 5 alpha-reductase for treatment of prostate cancer, we have studied the topography of the 5 alpha-reductase active site (5 alpha-R) and of the related androgen (RA) and progesterone (RP) receptors in the region complementary to C.6 of progesterone. To this end we have determined the total structures of 17 alpha-acetoxy-6-methylene-4-pregnene-3,20-dione (VII; R = H) and of 17 beta-hydroxy-6,6-ethylene-4-androsten-3-one (VIa) by X-ray crystal structure analysis and, using these data, have developed Newman projections of the 6 alpha-Me, 6 beta-Me, 6-methylene and 6,6-ethylene derivatives of progesterone. From them we have developed a Newman projection of a composite model formed from steroids (V), (VI), (VIIIa) and (VIIIb). This is shown in Fig. 4 and illustrates the relative conformations of these substituents around C.6. From there we proceeded to receptor-binding studies. Our results led to the conclusion that androgen receptor, (RA), takes up preferred but different conformations when bound to testosterone (T) and to 17 beta-hydroxy-5 alpha-androstan-3-one (5 alpha-dihydrotestosterone, DHT), respectively, and that the resulting steroid-receptor complexes bind preferentially to different chromatin acceptor sites. We have therefore used the convention RT and RDHT in place of RA as appropriate. Working on the assumption that binding affinities reflect spatial contours, we have developed comparative silhouettes for the 5 alpha-R, RP and RDHT protein binding sites complementary to C.6 of the steroidal ligand. These data show that the 5 alpha-reductase active site is characterized by a hydrophobic pocket which specifically accommodates a 6-methylenic moiety and partially accommodates a 6 beta-methyl group. RDHT, in contrast, shows much less specificity and largely accommodates all the above substituents. Progesterone receptor differs in failing to accommodate 6,6-ethylene and 6 beta-methyl, with minimal accommodation of 6-methylene. It possesses a hydrophobic pocket skewed towards the alpha-face of the steroid, thereby allowing optimal binding of the 6 alpha-methyl substituent to the receptor. 6-Methylene-4-pregnene-3,20-dione (V) fails to bind significantly to androgen and progesterone receptors thereby supporting the postulate that its antiprostatic activity stems primarily from 5 alpha-reductase inhibition.     

NMR studies of the stereospecificity of 20 -hydroxysteroid dehydrogenase

We (3,4) previously observed the reduction of 21-dehydrocorticosteroids in the presence of 20β-hydroxysteroid dehydrogenase proceeded at a faster rate than the reduction of the corresponding corticosteroids. The presence of adjacent carbonyl groups suggested the possibility that the increased rate of reduction of the 20-one,21-a1 steroid analogs resulted from a lack of specificity of the enzyme 20β-hydroxysteroid dehydrogenase for either the aldehyde or ketone group. Nuclear magnetic resonance spectroscopy indicated that the angular methyl groups of the steroid were sensitive probes for the constituents on the basic steroid skeleton. The C₁₈ methyl resonance of 17,21-dihydroxy-4-pregnene-3,20-dione and 17-hydroxy-3,20-dioxo-4-pregnene-21-a1 were 0.722 ppm and 0.728 ppm respectively. The magnitude and sign of the change in chemical shift of the C₁₈ methyl resonance for the enzymatic products of 17,21-dihydroxy-4-pregnene-3,20-dione and 17-hydroxy-3,20-dioxo-4-pregnene-21-a1 (+0.135 ppm and +0.144 ppm respectively) were consistent with a stereochemical assignment of 20β-hydroxyl.