Conversion of 20alpha-hydroxy-4-pregnen-3-one to 20alpha-hydroxy-5alpha-pregnan-3-one and 5alpha-pregnane-3alpha, 20alpha-diol by rat anterior pituitary

³H-20α-hydroxy-4-pregnen-3-one was incubated with anterior pituitaries from proestrous rats. The $\text{in vitro}$ metabolic products, identified by reverse isotopic dilution and purification to constant specific activity, were 20α-hydroxy-5α-pregnan-3-one (23.0%) and 5α-pregnane-3α,20α-diol (11.4%). These are qualitatively the same metabolites which result from $\text{in vitro}$ incubation of 20α-hydroxy-4-pregnen-3-one with medial basal hypothalamus. 68.8% of the recovered radioactivity remained as 20α-hydroxy-4-pregnen-3-one. These three compounds accounted for all of the recovered radioactivity.     

Identification of 20 alpha-hydroxy-5 alpha-pregnan-3-one and 20 beta-hydroxy-5 alpha-pregnan-3-one in human pregnancy urine

20 beta-Hydroxy-5 alpha-pregnan-3-one and 20 alpha-hydroxy-5 alpha-pregnan-3-one were isolated and identified from a pool of urine collected from women in the third trimester of pregnancy. Following isolation by Sephadex LH-20 and HPLC, the identity of each compound was established by comparison of GC-MS data for the methyloxime-trimethylsilyl ethers with those for authentic standards.     

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.     

Structure determination of 3 beta, 17-dihydroxy-17 alpha-pregn-5-ene-20-one

The title compound was synthesized as part of an effort to determine the identity of an abnormal steroid metabolite present in the urine of a patient exhibiting pronounced gynecomastia. The X-ray investigation of the synthesized compound showed that the 20-carbonyl of the 17 alpha oriented side chain lies under the D ring, and does not participate in hydrogen bonding in the crystal lattice. This conformation appears to be stable and sufficiently shielded that it is unlikely to make a major contribution to possible protein interactions.     

Synthesis of fluorinated 3β-hydroxypregn-5-en-20-one derivatives

Treatment of the unstable 3β-hydroxy-20, 20-dimethoxypregn-5-ene 3-acetate with acetic anhydride at reflux temperature gave a mixture of 3β-hydroxy-20-methoxypregna-5, 17(20)-diene and 3β-hydroxy-20-methoxypregna-5, 20-diene 3-acetates. Fluorination of this mixture with perchloryl fluoride afforded after fractionated crystallization 3β-hydroxy-17-fluoro-20-methoxypregna-5, 20-diene 3-acetate. Acid hydrolysis of the reaction mixture and subsequent Chromatographic separation led to 3β-hydroxy-17-fluoropregn-5-en-20-one 3-acetate and 3β-hydroxy-21-fluoropregn-5-en-20-one 3-acetate. 3β-Hydroxy-17-fluoro-20-methoxypregna-5, 20-diene 3-acetate did not react further with perchloryl fluoride even under forcing conditions. Fluorination of 3β-hydroxy-20-(N-ethyl benzylamino)-pregna-5, 17(20)-diene gave 3β-hydroxy-17, 21-difluoro-pregn-5-en-20-one, exclusively.     

Conversion of 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one to delta 16-C19 steroids by the reconstituted delta 16-C19-steroid synthetase system

The substrate specificity of the reconstituted delta 16-C19-steroid synthetase system, which catalyzes the formation of 5,16-androstadien-3 beta-ol or 4,16-androstadien-3-one from pregnenolone or progesterone, respectively, was studied. The reconstituted system consisted of a partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5 and NADH-cytochrome b5 reductase all from pig testicular microsomes. It was found that 5 alpha-reduced C21 steroids such as 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one can be substrates for the enzyme system, resulting in the formation of 5 alpha-androst-16-en-3-one, 5 alpha-androst-16-en-3 alpha-ol and 5 alpha-androst-16-en-3 beta-ol, respectively. The results suggest that 5 alpha-reduced delta 16-C19 steroids might be synthesized from pregnenolone and progesterone via 5 alpha-reduced C21 steroids as intermediates. The pathways would bypass 5,16-androstadien-3 beta-ol and 4,16-androstadien-3-one which have been assumed as obligatory intermediates in the formation of 5 alpha-reduced delta 16-C19 steroids from pregnenolone and progesterone.     

Radioimmunoassay of 3 alpha-hydroxy-5 alpha-pregnan-20-one in rat and human plasma

A radioimmunoassay for measuring 3 alpha-hydroxy-5 alpha-pregnan-20-one in plasma has been developed. Polyclonal antibodies were raised in rabbits against 3 alpha-hydroxy-20-oxo-5 alpha-pregnan-11 alpha-yl carboxymethyl ether coupled to bovine serum albumin. 3 alpha-Hydroxy-5 alpha-pregnan-20-one was purified from either extracts of plasma by high-performance liquid chromatography. These antibodies were then used for the radioimmunoassay of this centrally active progesterone metabolite in rat and human plasma. 3 alpha-Hydroxy-5 alpha-pregnan-20-one was detected in plasma from female rats on the day of estrus (2.0 to 9.3 ng/ml) and in the plasma of women during the luteal phase of the menstrual cycle at levels ranging from 0.25 to 2.5 ng/ml. The latter was highly correlated with plasma progesterone levels.     

Microbial transformation of 5alpha,6alpha-epoxy-3beta-hydroxy-16-pregnen-20-one by Trichoderma viride

Fermentation of 5alpha,6alpha-epoxy-3beta-hydroxy-16-pregnen-20-one (4) with Trichoderma viride under aerobic condition yielded 3beta,5alpha,6beta-trihydroxy-16-pregnen-20-one (5) and 3beta,5alpha,6beta,15beta-tetrahydroxy-16-pregnen-20-one (6). Each microbial metabolite was characterized by spectroscopic methods. Compounds 6 and 3beta,5alpha,15beta-trihydroxy-16-pregnen-6,20-dione (7) are reported for the first time.     

A new approach to the synthesis of 3β,23-diacetoxy-24-nor-5-cholene from 3β,21-diacetoxy-5-pregnen-20-one

Synthesis of 3β,23-diacetoxy-24-nor-5-cholene in six steps from 3β,21-diacetoxy-5-pregnen-20-one has been achieved by a new approach. The method would allow to label the side chain of a bile acid at carbon-atom 21.     

Significance of 3alpha, 17 alpha-dihydroxy-5beta-pregnane-20-one in the diagnosis of congenital adrenal hyperplasia.

The 3alpha-17alpha-dihydroxy-5beta-pregnane-20-one (17-hydroxy-pregnanolone) excretion was found by gas chromatography to be as high as the pregnanetriol in urine of 4 virilising CAH female and 2 male patients. According to our results the significance of the determination of 17-hydroxy-pregnanolone is equal to that of the pregnanetriol in the diagnosis of adrenal hyperplasia. As the 17-hydroxy-pregnanolone is demonstrable after mild acid hydrolysis, too, it can be applied independently from the pregnanetriol determination. In five out of the six patients we found the excretion of the pregnanediol to be high, too. Its value reached, irrespective of sex, the characteristic quantity of the woman's luteal phase, so it undoubtedly originated from the adrenal cortex. We dealt shortly with the explanation of high pregnanediol excretion.     

The mechanism investigation in substitution of 21-bromo-3alpha-hydroxyl-3beta-methoxymethyl-5alpha-pregnan-20-one with nucleophiles

A mechanistic study on the nucleophilic substitution of a strictly geometric 21-bromo-3alpha-hydroxyl-3beta-methoxymethyl-5alpha-pregnan-20-one was described. Reaction of the alpha-bromoketone with excess lithium imidazole followed by the addition of extra bases including n-butyllithium, methyllithium, lithium piperidine, and lithium pyrrolidine provided unexpected alpha-nucleophilic carbonyl adducts that derived from strong base. Data from HPLC and proton NMR suggested an epoxide as the intermediate. Two possible reaction pathways were proposed for the nucleophilic substitution reaction. One pathway is the normal SN2 substitution reaction, directly provided the imidazoly product without the formation of the unexpected alpha-substituted products. The other pathway went through an epoxide intermediate, in which imidazole anion or the strong bases added would attack from the less hindered site of the epoxide to give the substitution product.     

Conversion of 15α-hydroxy-11-oxo-progesterone to 3β,11α,15β-trihydroxy-5-pregnen-20-one

3β,11α,15β-Trihydroxy-5-pregnen-20-one, an intermediate required for the synthesis of the oogoniols, has been prepared from 15α-hydroxy-11-oxo-progesterone in an overall yield of 16%. The three isomers (at C-11, C-15) of the pregnene were also prepared.     

An efficient synthesis of 3 alpha-hydroxy-5 alpha-pregna-9(11),16-diene-20-one

3 alpha-Hydroxy-5 alpha-pregna-9(11),16-diene-20-one (6) was synthesized for the first time from the commercially available 16,17 alpha-epoxy-3 beta-hydroxy-5-pregnen-20-one(1) in several steps. The key step involves a remote chlorination reaction utilizing m-iodobenzoate group as a free-radical guide.     

Comparative facilitation and inhibition of lordosis in the guinea pig with progesterone, 5-alpha-pregnane-3,2-dione, or 3-alpha-hydroxy-5-alpha-pregnan-20-one

The major 5α-reduced metabolites of progesterone tentatively identified in neural tissue of the guinea pig were evaluated in this species for their ability to facilitate and inhibit lordosis responses of spayed females after estradiol benzoate (EB) pretreatment. 5α-Dihydroprogesterone was found to be an effective facilitative agent, but at doses of 0.05-0.3 mg administered at time intervals from 12–60 hr after estradiol, it was not as potent as progesterone. The steroids 3α-hydroxy-5α-pregnan-20-one and 5β-pregnane-3,20-dione, evaluated at only one dose level (0.18 mg) and at one time interval after estradiol (36 hr), were found to have moderate facilitative effects, but they were not as effective as 5α-dihydroprogesterone.The inhibitory influences of the metabolites studied were found to be weak relative to progesterone when given at doses of 0.6 mg 1 hr after EB. However, when 5α-dihydroprogesterone was given at a higher dose (3.6 mg) it was then found to be an effective inhibitor of the lordosis response. The results indicate that this metabolite has behavioral influences similar to those of progesterone for both facilitation and inhibition of estrus. It was suggested that the superior potency of injected progesterone may be due to mechanisms of bioavailability, including relative solubility differences of the two steroids when administered subcutaneously.     

The reduction of 5 alpha-cholestan-3-one and 5 beta-cholestan-3-one by some boranes and hydroborates.

This paper describes the high yield synthesis of 5 alpha-cholestan-3 alpha-ol and 5 beta-cholestan-3 beta-ol by the reduction of 5 alpha- and 5 beta-cholestan-3-one using using potassium and lithium-tri-sec-butyl-hydroborates as reducing reagents. Attempts to obtain the same alcohols using other bulky boranes resulted in the equatorial products.