Synthesis of 18,19-dihydroxycorticosterone.

A four-step synthesis of 18,19-dihydroxycorticosterone 5c, starting with 19,21-dihydroxy-3,20-dioxopregn-5-ene-18,11 beta-lactone-di-(ethylene ketal) 2, is presented. Reduction of 2 with sodium aluminum bis-(methoxyethoxy)hydride gave 11 beta,18,19,21-tetrahydroxy-pregn-5-ene-3,20-dione-di-(ethylene ketal) 3a. Acetylation furnished the corresponding 18,19,21-triacetate 3b, which on treatment with a mixture of perchloric and acetic acids gave 18,19-dihydroxycorticosterone 18,19,21-triacetate 4b. Mild saponification yielded the title compound which, on the basis of ir and nmr spectra, exists as one C-20 isomer of the hemiacetal structure 5c. Periodate oxidation of 5c gave the expected 11 beta, 19-dihydroxy-3-oxoandrost-4-ene-17 beta, 18-carbolactone 6b.

18,19-Dihydroxydeoxycorticosterone, a new metabolite produced from 18-hydroxydeoxycorticosterone by cytochrome P-450(11) beta. Chemical synthesis and structural analysis by 1H NMR.

A new metabolite was produced from 18-hydroxydeoxycorticosterone by the cytochrome P-450(11) beta linked hydroxylase system purified from bovine adrenocortical mitochondria. It was identified as 18,19-dihydroxydeoxycorticosterone by chemical synthesis on the basis of high-performance liquid chromatography, gas chromatography-mass spectrometry, and proton nuclear magnetic resonance (1H NMR) spectroscopy, and detailed structural analysis of it was performed by 1H NMR spectroscopy. The methylene protons at the C-19 position of the steroid were nonequivalent and coupled with each other, having a coupling constant of 10.6 Hz. These protons had different coupling constants, 6.7 and 3.4 Hz, for the hydroxy proton at the C-19 position. Due to these couplings, the signals of the methylene protons were observed around 3.9 ppm as two double doublets. The methylene protons at the C-21 position were also nonequivalent, having a coupling constant of 11.1 Hz. Coupling constants between these methylene protons and the hydroxy proton at the C-21 position were 8.2 and 4.2 Hz, respectively. These results indicate that both hydroxymethyl groups at the C-19 and C-21 positions do not freely rotate in chloroform solution. The signals of hydroxy protons at the C-19 and C-21 positions were found at 1.25 and 1.87 ppm, respectively, by means of decoupling of the corresponding methylene protons. The hydroxy proton at the C-18 position was found to scarcely couple with any proton. This fact suggests that this hydroxy group is linked to the C-20 position, making a hemiketal bridge between the C-18 and the C-20.

Affinity of 18,19-dihydroxydeoxycorticosterone and 18-hydroxy-19-nor-deoxycorticosterone to aldosterone receptor and their mineralocorticoid activity.

Affinity of 18,19-dihydroxydeoxycorticosterone (18,19-diOH-DOC) and 18-hydroxy-19-nor-deoxycorticosterone (18-OH-19-nor-DOC) to aldosterone receptor and their mineralocorticoid activity were evaluated. 18,19-DiOH-DOC (1 X 10(-6) M) did not show appreciable binding to the receptor and its relative potency as a mineralocorticoid was estimated to be less than 1/8,000 of that of deoxycorticosterone (DOC). 18-OH-19-nor-DOC bound to the receptor with an affinity similar to that of 18-hydroxydeoxycorticosterone (18-OH-DOC) or spironolactone. Its sodium retaining activity was 0.04 times as great as that of DOC and similar to that of 18-OH-DOC.

Corticosteroid regulation of electrolytes.

It is proposed that sodium and potassium are regulated by varying the amounts of aldosterone, DOC, 18 OH-DOC, and 16 alpha 18 dihydroxy 11 deoxycorticosterone secreted in response to the nutritional load. The first two steroid hormones are for high potassium and the second two for low potassium intake. The nutritional load acts on potassium regulators primarily through its affect on serum potassium. The first and third steroids are proposed for low sodium intake.

19-Hydroxylation of 18-hydroxy-11-deoxycorticosterone catalyzed by cytochrome P-45011 beta of bovine adrenocortex.

When 18-hydroxy-11-deoxycorticosterone was incubated with adrenocortical mitochondria fortified by adding exogenously adrenodoxin reductase and adrenodoxin in the presence of an NADPH-generating system, the steroid was converted to two products which were separated by high performance liquid chromatography. The retention time of one product was identical with that of 18-hydroxycorticosterone, an expected product of 11 beta-hydroxylation, whereas the retention time of the other was not coincident with those of any other corticosteroids examined. Production of the unidentified substance was also shown when 18-hydroxy-11-deoxycorticosterone was incubated with purified cytochrome P-45011 beta and its electron transport system. The ratio of the two products was always constant, even when various incubation conditions were employed with regard to reaction time, the substrate concentration, and the cytochrome concentration. Structural determination of the unidentified product was conducted by mass spectrometry and 1H NMR spectrometry. The results of these analyses indicated that the substance was 18,19-dihydroxy-11-deoxycorticosterone, a hitherto unreported corticosteroid.

18-substituted steroids. Part 4. A chemical synthesis of 3 alpha,18,21-trihydroxy-5 beta-pregnan-20-one 18--20-hemiacetal (18-hydroxy-tetrahydro-DOC).

3 alpha,18,21-Trihydroxy-5 beta-pregnan-20-one 18 leads to 20-hemiacetal (18-hydroxy-tetrahydro-DOC) has been prepared from 3 alpha-acetoxy-5 beta-pregnan-20-one by reduction to the 20 beta-alcohol, application of the 'hypoiodite' reaction [Pb(OAc)4-I2-hv] with subsequent steps leading to the 18-hydroxy-20-ketone (as hemiacetal), and C-21 acetoxylation [Pb(OAc)4] followed by hydrolysis.

Role of 18-hydroxy-11-deoxycorticosterone and 16 alpha, 18-dihydroxy-11-deoxycorticosterone in hypertension.

Excess secretion of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) occurs in more than 10% of hypertensive patients with suppressed plasma renin activity, but it also is reported to occur in essential hypertension without impairment of the renin system. Preliminary studies measuring plasma 18-OH-DOC by radioimmunoassay support the idea that 18-OH-DOC secretion is elevated in some patients with essential hypertension. Interpretation of these data must take into account endogenous ultradien and circadian variations in plasma 18-OH-DOC, however. 18-OH-DOC serves as a precursor of another steroid secretory product. Conversion of labeled 18-OH-DOC to a new structure, 16alpha, 18-dihydroxy-11-deoxycorticosterone (16alpha, 18-diOH-DOC), was demonstrated to be greatly accelerated by the adrenal tissue in low-renin patients as compared with those with normal adrenal tissue (70 to 80% versus 15% conversion). Hypersecretion of 16alpha, 18-diOH-DOC occurred in each. This steroid exerted no effect on sodium metabolism in adrenalectomized rats or in the toad bladder assay, but it markedly enhanced activity of subthreshold doses of aldosterone in reducing sodium excretion in urine of adrenal-ectomized rats. Because of the unique activity of this steroid, we have concluded that excessive 16alpha, 18-diOH-DOC secretion may be important in the genesis of suppressed renin in some patients with hypertension.

Adrenocortical factors in hypertension. I. Significance of 18-hydroxy-11-deoxycorticosterone.

Low renin essential hypertension and the syndrome of mineralocorticoid excess have two features in common, low plasma renin activity and volume-sensitive hypertension. The proposal that both disorders share a common mechanism--because of the ability of agents that inhibit or antagonize the adrenocortical secretion to lower blood pressure in the low renin hypertensive group--appears to be based on a circular argument. The beneficial effect of removal or neutralization of the adrenocortical contribution only constitutes evidence for volume-dependency or sensitivity, which is how the low renin group is defined. Any measure that blocks a component of the normal homeostatic chain for the maintenance of extracellular and intravascular volume including the adrenal cortex would be expected to have a beneficial effect in volume-sensitive hypertension. Evidence for an adrenal factor in low renin hypertension must rest on the isolation of an active substance that reproduces the effect when readministered. 18-Hydroxy-11-deoxycorticosterone (18-OH-DOC) does not meet these criteria. It is not significantly increased in experimental hypertension and, although its overproduction in unselected low renin essential hypertensive patients remains controversial, the magnitude of the reported elevations is insufficient in relation to the low biologic activity of the steroid to account for a significant effect. Apart from its increase in the 17alpha-hydroxylase defect, 18-OH-DOC is increased in primary aldosteronism and may also be an indicator of a histologic variant of the aldosteronoma. On the basis of a large body of evidence showing parallelism between the 11beta- and 18-hydroxylase functions of the fasciculata zone, we have proposed that both enzymic functions are functionally related and may involve the same enzyme protein and catalytic site. According to this view, the secretion of 18-OH-DOC would have no special significance of its own but would be an obligatory consequence of the secretion of fasciculata zone corticosterone.

New mineralocorticoids and adrenocorticosteroids in hypertension.

Alterations in steroidogenesis have been demonstrated in experimental and human hypertension. It is highly likely that increased secretion of the nonaldosterone mineralocorticoid deoxycorticosterone (DOC) and 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) may initiate or perpetuate hypertension, or both. It is possible that 16 beta-hydroxydehydroeplandrosterone (16beta-OH-DHEA) directly induces the hypertensive process in animals. The significance of the findings of increased secretion of 16 alpha, 18-dihydroxy-11-deoxycorticosterone (16alpha, 18-diOH-DOC) and dehydroepiandrosterone sulfate (DHEA-S) cannot now be appreciated. Neither has been examined experimentally for its ability to induce hypertension, and the former compound is not a mineralocorticoid. It does possess the curious property of increasing mineralocorticoid activity of other steroids, by altering either their metabolism or mode of action. Variations in the mineralocorticoid hypertensive syndrome or, more aptly, the steroid hypertensive syndrome could account for the hypertension in a substantial portion of patients with reduced plasma renin activity.

Microbiological synthesis of 16alpha, 18-dihydroxydeoxycorticosterone.

16Alpha, 18-Dihydroxydeoxycorticosterone (16alpha, 18-dihydroxy-DOC) (1) and 1, 2-3H-16alpha, 18-dihydroxy-DOC (1,2-3H-16alpha, 18-dihydroxy-DOC) of high specific activity were obtained in good yield by microbiological hydroxylation of 18-OH-DOC and 1,2-3H-18-OH-DOC by Streptomyces roseochromogenus (ATCC 13400). The identity of the fermentation product to human adrenal produced 16alpha, 18-dihydroxy-DOC was established by chromatographic studies, derivative formation and gas-liquid chromatography. Yield of the product was about 30% of sutstrate and, allowing for losses in recovery, about 60% of the substrate 18-OH-DOC was converted to this product. A second product of fermentation isolated in lower yield appeared to be a dimer of 16alpha, 18-dihydroxy-DOC found in the acidic conditions of the fermentation. This method of synthesis of 16alpha, 18-dihydroxy-DOC is a practical way to make large quantities of the compound for further study of its possible role in human and experimental hypertension.