Corticosteroid side chain oxidations--II. Metabolism of 20-dihydro steroids and evidence for steroid acid formation by direct oxidation at C-21.

Rabbits have been injected with 4-14C-labelled progesterone, deoxycorticosterone and corticosterone and the corresponding 20 beta-3H-reduced steroids (20-dihydro steroids) in order to compare the influence of oxidation at C-20 on the excretion of steroid acids. Both 20 beta-reduced progesterone and deoxycorticosterone were extensively oxidized at C-20 and metabolized to 20-oxo-21-oic acids devoid of tritium. A small proportion of the acidic metabolites of [20 beta-3H]dihydro deoxycorticosterone retained tritium. By contrast the majority of the metabolites of [20 beta-3H]dihydro corticosterone were tritiated and [11 beta,20 beta-3H]-dihydroxy-4-pregnene-3-one-21-oic acid was identified as a major acidic metabolite. These results indicate that the presence of a 11 beta-hydroxyl in 20 beta-dihydro corticosterone inhibits oxidation at C-20 and provides evidence for the direct oxidation of this corticosteroid at C-21 in this species.     

Side-chain cleavage and C-20 ketone reduction of hydrocortisone by a natural isolate ofChroococcus dispersus

A unicellular cyanobacterium,Chroococcus dispersus (Keissl.) Lemmermann, was isolated from paddy-field and tested in biotransformation experiments of hydrocortisone (compound 1). This strain has not been previously examined for steroid substance modification. Fermentation was carried out in BG-11 medium supplemented with 0.05% substrate at 25 °C for seven days incubation. The metabolites were chromatographically purified and characterised using spectroscopic methods. The fermentation yielded 11β,17α,20β,21-tetrahydroxypregn-4-en-3-one (compound 2), 11β,17β-dihydroxyandrost-4-en-3,17-dione (compound 3), and 11β-hydroxyandrost-4-en-3,17-dione (compound 4). Bioreaction characteristics observed were 20-ketone reduction for accumulation of compound 2 and side chain degradation of the substrate to give compounds 3 and 4. Time course study showed the accumulation of the product 2 from the second day of the fermentation and product 3 as well as product 4 from the third day. All the metabolites reached their maximum concentration in seven days. Aeration and continuous light or light duration (16/8 hours light/dark) have no effect on the transformation yield. Optimum concentration of the substrate, which gave maximum bioconversion efficiency, was 0.5 mg ml^?1 in the transformation experiment. Growth was not influenced by the addition of steroid substrate. Biotransformation was completely inhibited when steroid concentration was above 2.0 mg ml^?1.     

Stereoselective reduction of C-2 substituted steroid C-3 ketones with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride and sodium borohydride

The effect of C-2 substitution on the stereoselective reduction of steroid C-3 ketones with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride and sodium borohydride was investigated. The C-2 mono- and di-substituted chloro and methyl derivatives were predominantly reduced to one of the epimeric alcohols. The 2 alpha-chloro and 2 alpha-methyl derivatives of 17 beta-acetoxy-5 alpha-androstan-3-one undergo stereoselective reduction with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride to the axial (3 alpha) alcohol as observed in the unsubstituted compound, whereas sodium borohydride gives predominantly the equatorial (3 beta) alcohol. The 2 beta-chloro, 2 beta-methyl, 2,2-dichloro, and 2,2-dimethyl derivatives are reduced predominantly to the equatorial (3 beta) alcohol by both reagents.     

The radioimmunoassay of steroid glucuronides. The oestrogen C-3 glucuronides as haptens.

Antisera were prepared against three related oestrogen ring-A glucuronides, oestrone 3-glucuronide, oestradiol 3-glucuronide and oestriol 3-glucuronide. The corresponding 6,7-3H-labelled conjugates were synthesized as radioligands and the cross-reactions of the antisera against ring-A oestrogen glucuronides and other steroid conjugates were examined. The specificity of the antiserum against oestriol 3-glucuronide was compared with that raised against oestriol 16alpha-glucuronide, and the measurement of the former conjugate in late-pregnancy urine is discussed.     

Synthesis of C-19 deuterium labelled steroids.

Preparation of the synthetically useful steroid intermediate 19-d3-androst-4-ene-3,17-dione (Ia) together with its 19-d2-(Ib) and 19-d1-(Ic) analogues is described. The conditions and work-up of the synthesis have been designed to eliminate tedious chromatographic separation and purification steps thus enabling decigrams of material to be conveniently prepared using standard laboratory apparatus. The deuterium label in the C-19 angular methyl group is inert to normal chemical exchange processes, thus offering the opportunity for synthesis of more complex, biologically active, stable labelled steroids, whose metabolism can be studied mass spectrometrically.     

Synthesis of C-2 and C-4 deuterium-labeled estradiol-17 beta

Estradiol-17 beta labeled with deuterium in the positions 2 or 4 can be prepared from 2-chloromercurio-1,3,5(10)-estratriene-3,17 beta-diol 3-methyl ether 17-acetate or 4-chloromercurio-1,3,5(10)-estratriene-3,17 beta-diol, respectively, in refluxing CH3COO(2)H/(2)H2O. The same reaction performed on 4-acetoxymercurio-1,3,5(10)-estratriene-3,17 beta-diol afforded 2,4-dideuterio-estradiol-17 beta in good yields.     

Synthesis and biological evaluation of C-3'NH/C-10 and C-2/C-10 modified paclitaxel analogues

Concurrent modifications on the C-3'NH/C-10, and C-2/C-10 positions on paclitaxel were carried out as a way of investigating possible synergistic effects. The biological activities of these analogues were evaluated in both a microtubule assembly assay and human ovarian cancer (A2780) and prostate cancer (PC3) cytotoxicity assay. In some cases the doubly modified analogues were more active than would have been predicted based on the activity of the singly modified analogues, indicating probable synergistic effects.     

Isolation of urinary C-20 alpha- and C-20 beta-hydroxy-C21-steroid metabolites in cases of congenital adrenal hyperplasia, postpubertal virilizing syndrome and polycystic ovary syndrome.

The proportion of 20 alpha/20 beta-epimers of the urinary C21-steroid metabolites was estimated in normal volunteers and in patients with congenital adrenal hyperplasia (CAH), postpubertal virilizing syndrome (PVS) and polycystic ovary syndrome (POS). In the normal individuals 20 alpha- and 20 beta-epimers of 5 beta-pregnane-3 alpha, 17 alpha, 20-triol (pregnanetriol) and 5-pregnene-3 beta, 17 alpha, 20-triol (5-pregnenetriol) were measured. In the other case 20 alpha- and 20 beta-epimers of the characteristic, representative metabolites were also investigated. In 26 of 27 normal persons and in all the patients with normotensive CAH, PVS and POS the 20 beta-epimer comprised 0-10% of the total pregnanetriol. The 20 beta-epimer of 5-pregnenetriol was found in only one normal case (6% of the total). The percentage of 20 beta-epimers of 3 alpha, 17 alpha, 20-trihydroxy-5 beta-pregnan-11-one, 5 beta-pregnane-3 alpha, 11 beta, 17 alpha, 20-tetrol (in the normotensive CAH, PVS and POS) and 5 alpha- or 5 beta-pregnane-3 alpha, 17 alpha, 20, 21-tetrol (in the hypertensive CAH) varied from nil to 76%. The effect of functional groups and the stereochemistry of the molecule on the direction of C-20-keto group reduction is discussed; the existence of additional factors determining this reduction in certain pathological conditions is suggested.     

Synthesis of hydantocidin and C-2-thioxo-hydantocidin

Hydantocidin, a naturally occurring strong herbicide, was synthesized in an overall yield of 35.2%, with the accompanying 1'-epi-hydantocidin in overall 9.6% yield from 2,3-O-isopropylidene-D-ribono-1,4-lactone. C-2-thioxo-hydantocidin and its spiro-epimer were also synthesized in an overall yield of 14.4% and 8.5%, respectively.