The 21-aminosteroid inhibitors of lipid peroxidation: reactions with lipid peroxyl and phenoxy radicals

The 21-aminosteroids U74006F and U74500A have been examined for their ability to scavenge the lipid peroxyl (LOO.) and phenoxy (PhO.) radicals. Lipid peroxidation was followed by measuring the formation of linoleic acid hydroperoxide (LOOH; 18:200H) from linoleic acid during incubations in methanol at 37 degrees C. Initiation of lipid peroxidation was by the radical generator 2,2'-azobis(2,4-dimethylvaleronitrile; AMVN), which under the conditions employed, initiated LOOH formation at a constant rate of 22 microM/h with a kinetic chain length of 21. Alpha-tocopherol (alpha TC) nearly completely blocked the chain reaction by scavenging LOO., reducing its formation to that essentially attributable to initiation alone. The average inhibition rate constant kinh for alpha TC at 37 degrees C was calculated as 4.9 x 10(5) M-1 sec-1. U74006F or U74500A also inhibited LOOH formation, reducing its rate to a constant fraction of control in a concentration dependent manner. U74500A was a more potent scavenger of LOO. than U74006F; however, both compounds were considerably less potent than alpha TC based upon their respective kinh's at 37 degrees C. Similarly, alpha TC, U74006F and U74500A scavenged PhO.. As seen with LOO. scavenging, alpha TC was orders of magnitude more reactive toward PhO. than either 21-aminosteroid as judged by their respective second order rate constants (k2). Both U74006F and U74500A were degraded during their reaction with LOO. or PhO. to as yet uncharacterized product(s). The data indicate that while the 21-aminosteroids can scavenge lipid radicals, their activity in this regard is less than expected based upon their ability to inhibit iron dependent lipid peroxidation.     

Evidence for 21-aminosteroid association with the hydrophobic domains of brain microvessel endothelial cells.

Studies were conducted to demonstrate 21-aminosteroid distribution into the hydrophobic or lipid domains of biological membranes, a presumed site at which these compounds inhibit lipid peroxidation. Bovine brain microvessel endothelial cells (BMECs) were labeled with diphenylhexatriene fluorophores and interactions with cell membranes characterized with fluorescence anisotropy and lifetimes. Two 21-aminosteroids (U-74500A and U74006F) were shown to preferentially alter the fluorescence anisotropy and lifetime parameters of the diphenylhexatriene probe distributing into membranes throughout the BMECs. Little or no effect of the compounds was observed on the fluorescence parameters of the probe localized on the surface of BMEC plasma membranes. By contrast, cholesterol used as a positive control substantially altered the fluorescence parameters of BMECs labeled with either diphenylhexatriene probe. Results suggest 21-aminosteroid-induced changes in the molecular packing order and drug: fluorescent probe interactions in membrane hydrophobic (or lipid) domains throughout the BMEC. Concentrations of 21-aminosteroids altering the fluorescence parameters of diphenylhexatriene labeled BMECs correspond to those concentrations of 21-aminosteroids effective in vitro in inhibition of lipid peroxidation.     

Microbial conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione acetates by Nocardioides simplex VKM Ac-2033D

The conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione 21-acetate (I) and 17,21-diacetate (VI) by Nocardioides simplex VKM Ac-2033D was studied. The major metabolites formed from I were identified as pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV). Pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione (III) and pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V) were formed in minorities. Biotransformation products formed from VI were pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17,21-diacetate (VII), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII), pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V). The conversion pathways were proposed including 1(2)-dehydrogenation, deacetylation, 20beta-reduction and non-enzymatic migration of acyl group from position 17 to 21. The conditions providing predominant accumulation of pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) from I and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII) from VI in a short-term biotransformation were determined.     

High-performance liquid chromatographic determination of a 21-aminosteroid antioxidant in plasma

A novel 21-aminosteroid, 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-16α-methylpregna-1,4,9(11)-triene-3,20-dione monomethanesulfonate (I), is under development for the treatment of central nervous system injury in humans. This report describes a reversed-phase high-performance liquid chromatographic (RP-HPLC) method using ultraviolet detection at 254 nm for the determination of I in plasma with low nanogram per milliliter sensitivity. Plasma was deproteinated by mixing with acetonitrile and centrifuging, and I was extracted from the supernatant with disposable bonded-phase columns. The extracts were chromatographed on an octylsilane bonded-phase HPLC column with an acetonitrile—water mobile phase containing triethylammonium acetate, pH 5. Quantification was accomplished by peak-height ratio analysis using an analogue of I as the assay internal standard. The method was suitable for the determination of I following a 30 mg/kg intraperitoneal dose in the rat.     

Inhibition of S-(1,2-dichlorovinyl)-L-cysteine-induced lipid peroxidation by antioxidants in rabbit renal cortical slices: Dissociation of lipid peroxidation and toxicity

Precision-cut, rabbit renal slices were used to examine the effects of three novel antioxidants (U-74006, U-74500, and U-78517) on S-(1,2-dichlorovinyl)-L-cysteine (DCVC)-induced lipid peroxidation and toxicity. Slices exposed to DCVC showed a dose- and time-dependent increase in lipid peroxidation (TBARS) and a decrease in cellular viability, as evidenced by the loss of intracellular potassium, during the course of a 3 hour incubation. Subsequent studies employed DCVC concentrations of 100 μM. Microemulsion formulations of U-78517, U-74500, and U-74006 (100 μM) inhibited DCVC-induced lipid peroxidation by 100±, 50±, and <5% (not significant), respectively. However, none of these antioxidants had a significant effect on DCVC-dependent cytotoxicity, as indicated by intracellular potassium release. The effects of U-78517, the most potent of the three antioxidants, were similar to those observed with two model antioxidants, diphenyl-p-phenylenedi-amine (DPPD) and the iron chelator, deferoxamine. Aminooxyacetic (AOAA), an inhibitor of renal cysteine conjugate β-lyase, had only a minimal effect on DCVC-induced lipid peroxidation, and no effect on toxicity. These data represent the first report of DCVC-induced lipid peroxidation in rabbit renal cortical slices, a system which has been widely used to investigate mechanisms of nephrotoxicity, including that induced by DCVC. Our results demonstrate that DCVC-induced lipid peroxidation in renal slices can be inhibited by a variety of antioxidant compounds operating by different mechanisms. Because inhibition of lipid peroxidation had minimal effect on DCVC-dependent cytotoxicity, the data suggest that DCVC-induced lipid peroxidation is not a major mechanism in the cytotoxicity induced by this compound.     

Tirilazad Mesylate Protects Vitamins C and E in Brain Ischemia-Reperfusion Injury

Brain concentrations of the antioxidant vitamins C and E decreased following unilateral carotid occlusion and reperfusion for 2 or 24 h in gerbils. Administration of the 21-aminosteroid inhibitor of lipid peroxidation, tirilazad mesylate (U74006F), prevented the decrease in level of both of these vitamins following 2 h of reperfusion. After 24 h of reperfusion, however, alpha-tocopherol (vitamin E) continued to be protected, but ascorbic acid (vitamin C) showed a pronounced decrease in content. The changes in concentrations of these vitamins are consistent with U74006F acting to inhibit peroxidation in the CNS by scavenging of lipid peroxyl radicals and suggest that, in the presence of this agent, injury-induced depletion of ascorbic acid may occur without irreversible tissue damage.     

Inhibition of the Iron-Catalysed Formation of Hydroxyl Radicals by Nitrosouracil Derivatives: Protection of Mitochondrial Membranes Against Lipid Peroxidation

A new series of metal ligands containing the 1,3-dimethyl-6-amino-5-nitrosouracil moiety has been synthesized and they have been studied as potential inhibitors of iron-dependent lipid peroxidation. For this purpose, these new derivatives have been tested in the Fenton induced deoxyribose degradation assay, which allows a quantitative measurement of their inhibitory effect towards hydroxyl radical generation. When iron(II) is complexed by these ligands, a strong inhibition of deoxyribose degradation is observed, especially in the case of tris-[2-(1,3-dimethyl-5-nitrosouracil-6-yl)aminoethyl] amine (5). This inhibitory effect is clearly related to a specific complexation of iron(II) and is not due to the direct scavenging of hydroxyl radical by the ligand. Inhibition of the iron mediated Fenton reaction presumably results from inactivation of the reactivity of the metal center towards hydrogen peroxide. These derivatives, as well as long alkyl chain substituted nitrosouracils were evaluated in the protection of biological membranes against lipid peroxidation (induced by iron(II)/ dihydroxyfumaric acid and determined with the 2-thiobarbituric acid test). Ligand 5 inhibited lipid peroxidation at a rate similar to Desferal (desferrioxamine B) and slightly higher than bathophenanthroline sulphonate (BPS), which are respectively good iron(III) and iron(II) chelators. When covalently bound with a long alkyl chain, the increase of lipophilic character of the ligand allows its location near the mitochondrial membrane, where lipid peroxidation occurs. Lower concentrations (IC₅₀ = 4 μM) are then necessary to inhibit lipid peroxidation. This IC₅₀ concentration should be compared to those obtained for Trolox (IC₅₀ = 3 μM) or the 21-aminosteroid U74500A (IC₅₀ = 1 μM) described previously.     

In vitro anti-inflammatory activities of new steroidal antedrugs: [16alpha,17alpha-d] Isoxazoline and [16alpha,17alpha-d]-3'-hydroxy-iminoformyl isoxazoline derivatives of prednisolone and 9alpha-fluoroprednisolone

A series of new anti-inflammatory steroidal antedrugs with C-16,17-isoxazoline ring system were synthesized and their pharmacological activities were evaluated. We reported earlier that these compounds are promising antedrugs based on the results of 5-day rat croton oil ear edema assay. In the present study, most of these compounds showed high binding affinities to the glucocorticoid receptor of liver cytosol. 21-acetyloxy-9alpha-fluoro-11beta-hydroxy-3,20-dioxo-1,4-pregnadieno [16alpha,17alpha-d] isoxazoline (FP-ISO-21AC) and 11beta,21-dihydroxy-9alpha-fluoro-3,20-dioxo-1,4-pregnadieno [16alpha,17alpha-d] isoxazoline (FP-ISO-21OH) were found 5.0-, 5.3-fold more potent than prednisolone, respectively. Inhibitory effects of the antedrugs on the nitric oxide (NO) production were assessed using LPS-stimulated RAW 264.7 murine macrophage cells. All these steroidal antedrugs exhibited concentration-dependent inhibition of NO production, but their relative potencies were lower than prednisolone. In vitro metabolism study in rat plasma showed that FP-ISO-21AC and 21-acetyloxy-9alpha-fluoro-11beta-hydroxy-3,20-dioxo-1,4-pregnadieno [16alpha,17alpha-d]-3'-hydroxyiminoformyl isoxazoline (FP-OXIM-21AC) were hydrolyzed rapidly, with the half-lives of 2.1 and 4.2 min, respectively. The half-lives of FP-ISO-21OH and 11beta,21-dihydroxy-9alpha-fluoro-3,20-dioxo-1,4-pregnadieno [16alpha,17alpha-d]-3'-hydroxyiminoformyl isoxazoline (FP-OXIM-21OH) were 92.2 and 110.2 min, respectively.     

Iron-Melanin Interaction and Lipid Peroxidation: Implications for Parkinson''s Disease

The vulnerability of substantia nigral (SN) melaninized dopamine neurons to neurodegeneration in Parkinson's disease and the selective increases of iron and basal lipid peroxidation in SN indicate that iron-melanin interaction could be crucial to the pathogenesis of this disease. The present study describes, for the first time, the identification and characterization of a high-affinity (KD = 13 nM) and a lower affinity (KD = 200 nM) binding site for iron on dopamine melanin. The binding of iron to melanin is dependent on pH and the concentration of melanin. Iron chelators, U74500A, desferrioxamine, and to less extent 1,10-phenanthroline and chlorpromazine, but not the Parkinson-inducing neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, can inhibit the binding of iron to melanin and iron-induced lipid peroxidation. Although melanin alone diminishes basal lipid peroxidation in rat cortical homogenates, it can also potentiate that initiated by iron, a reaction inhibited by desferrioxamine. In the absence of an identifiable exogenous or endogenous neurotoxin in idiopathic Parkinson's disease, iron-melanin interaction in pars compacta of SN may be a strong candidate for the cytotoxic component of oxygen radical-induced neurodegeneration of melaninized dopamine neurons.     

Structure-activity relationships of steroid hormones on muscarinic receptor binding

A total of fifty steroidal compounds were tested for their inhibition on the binding of muscarinic receptor antagonist, [3H]quinuclidinyl benzilate ([3H](-)QNB), to the hypothalamic membranes prepared from male rats. Among the compounds tested, the active structures (with IC50 values less than or equal to 100 microM in parentheses) are: progesterone (40), 5 beta-pregnane-3,20-dione (40), deoxy-corticosterone (50), 5 beta-pregnane-17 alpha,21-diol-3,20-dione (30), 11-desoxy-17-hydroxycorticosterone (22), 17 alpha-hydroxyprogesterone (20), 5 beta-pregnan-17 alpha-ol-3,20-dione (24), 5 beta-androstane-3,17-dione (100), and 5 beta-dihydrotestosterone (100). By examining all the compounds tested, the following structure-activity relationship became apparent: (a) The ring A-reduced steroidal structures with a 5 beta-conformation were more potent than those with a 5 alpha-conformation; (b) 17 alpha-hydroxylation of the steroidal ring increased the steroid's inhibitory activity; (c) The C3 carbonyl group was essential for activity; (d) Reduction of the C3 carbonyl group or aromatization of the ring A abolished the steroid's inhibitory activity; (e) Oxidation of the C11 position of ring C resulted in a decrease or loss of inhibitory activity; and (f) Different modifications of the side chain of ring D by acetylation resulted in either an increase or a decrease in the inhibitory activity. The structure-activity relationship as revealed in this study might provide an insight for the synthesis of a steroidal molecule with a high affinity for the muscarinic receptor as well as for the search of a more potent and physiologically relevant steroidal metabolite possessing the ability to interact with the muscarinic receptor.     

New aromatic esters of progesterone as antiandrogens

The in vivo and in vitro antiandrogenic activity of four new progesterone derivatives: 4-bromo-17alpha-(p-fluorobenzoyloxy)-4-pregnene-3,20-dione 1,4-bromo-17alpha-(pchlorobenzoyloxy)-4-pregnene-3,20-dione 2, 4-bromo-17alpha-(p-bromobenzoyloxy)-4-pregnene-3,20-dione 3 and 4-bromo-17alpha-(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 5alpha-reductase with IC50 values of: 280 (1), 2.6 (2), 1.6 (3) and 114 microM (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.     

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.     

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.     

21-Acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione conversion by Nocardioides simplex VKM Ac-2033D

The conversion of 21-acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (I) by Nocardioides simplex VKM Ac-2033D was studied purposed selective production of its 1(2)-dehydroanalogues—value precursors in the synthesis of modern glucocorticoids starting from 9-hydroxyandrostenes. 21-Acetoxy-pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (II), pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (III) and pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (IV) were revealed as metabolites, and the structures were confirmed by mass spectrometry and ¹H nuclear magnetic resonance (NMR) spectroscopy. The metabolic pathways of I by N. simplex included 1(2)-dehydrogenation and deacetylation. The sequence of the reactions was shown to depend on the transformation conditions. The presence of both soluble and membrane associated steroid esterases in N. simplex was demonstrated using cell fractionation. Unlike inducible 1(2)-dehydrogenase, steroid esterase was shown to be constitutive. The conditions providing selective accumulation of II from I by whole N. simplex cells were determined.     

Synthesis of 4,19-disubstituted derivatives of DOC. Radioreceptor assay of some corticosteroid derivatives in human mononuclear leukocytes

Several new 4,19-substituted steroids and previously synthesized corticosteroids were assayed for affinity to type 1 receptors in human mononuclear leukocytes. 11 beta,19-epoxy-4,21-dihydroxypregn-4-ene-3,20-dione (2) was hydrogenated with Pd-C to yield a mixture of all four dihydro derivatives 5, accompanied by 4,21-diacetoxy-11 beta,19-epoxy-3-hydroxypregnan-20-one (6) and 21-acetoxy-11 beta,19-epoxy-4-hydroxypregnane-3,20-dione (7). With hot acetic + p-toluenesulfonic acid 5 underwent rearrangement to 21-acetoxy-11 beta,19-epoxypregn-5-ene-4,20-dione (8) Pd-C hydrogenation of 3,21-diacetoxy-5 beta,19-cyclopregna-2,9(11)-diene-4,20-dione (10) gave 3,21-diacetoxy-5 beta,19-cyclopregn-5-ene-4,20-dione (11) and the 9,11-dihydro derivative of the latter. Treatment of 10 with warm HCl furnished 19-chloro-4,21-dihydroxypregna-4,9(11)-diene-3,20-dione (13). Pd-C hydrogenation of its diacetate 14 afforded the 4,5-dihydro derivative 18, 19-chloro-21-acetoxypregn-9(11)-en-20-one (15), its 4-acetoxy derivative 16 and the 3,4-diacetoxy derivative 17. When tested in a radioreceptor assay in human mononuclear leukocytes the synthesized compounds showed only low relative binding affinities (RBA) to type 1 receptor, the highest being 0.72% for 13 (aldosterone = 100%). For comparison, other RBA in this system were: 19-noraldosterone, 20%; 18-deoxyaldosterone, 5.8%; 18-deoxy-19-noraldosterone, 4.7%; 18,21-anhydroaldosterone, 0.37%; 17-isoaldosterone, 7.6% and apoaldosterone, 4.3%     

Pregnanes in the root bark of Nerium odorum

Neridienone-A (12β-hydroxy-pregna-4,6,16-triene-3,20-dione), neridienone-B (20β,21-dihydroxy-pregna-4,6-diene-3,12-dione), 12β-hydroxy-pregna-4,6-diene-3,20-dione, 12β-hydroxy-pregn-4-ene-3,20-dione and 12β-hydroxy-16α-methoxy-pregna-4,6-diene-3,20- dione were obtained from the root bark of Nerium odorum.     

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.     

Coexistence of zinc and iron augmented oxidative injuries in the nigrostriatal dopaminergic system of SD rats

Clinical studies have demonstrated an excess of transition metals, including zinc and iron, in the substantia nigra (SN) of Parkinson's patients. In the present study, the neurotoxic effect of zinc was investigated using iron as a positive control. Addition of zinc or iron to brain homogenates increased lipid peroxidation. Zinc was less potent than iron in inducing lipid peroxidation. Coincubation with desferrioxamine prevented zinc- and iron-induced lipid peroxidation. Furthermore, glutathione (GSH), S-nitroso-N-acetylpenicillamine, or melatonin inhibited zinc-induced lipid peroxidation. The oxidative effect of zinc was further investigated in anesthetized rats. Seven days after intranigral infusion of zinc, lipid peroxidation was elevated in the infused SN, and dopamine content and tyrosine hydroxylase-positive axons were decreased in the ipsilateral striatum. Zinc was less potent than iron in inducing neurodegeneration in vivo. L-Buthionine-[S,R]-sulfoximine pretreatment (i.c.v.), which depletes cellular GSH levels, enhanced zinc-induced oxidative injuries in the nigrostriatal dopaminergic system. Moreover, simultaneous infusion of zinc and iron appeared to augment oxidative injuries in rat brain. Taken together, our results demonstrate that intranigral infusion of zinc caused degeneration of the nigrostriatal dopaminergic system in rat brain. Furthermore, coexistence of zinc and iron augmented oxidative injuries in rat brain. These findings indicate that both zinc and iron contribute to the etiology of Parkinsonism.     

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.     

Steroid derivatives

Mycobacterium flavum was used to effect the transformation of 16β-methyl-16,17-oxido-7β,11α-dihydroxypregn-4-ene-3,20-dione (I) and the final products were isolated and identified as 16β-methyl-16,17-oxido-7β,11α-dihydroxypregna-1,4-diene-3,20-dione (II) and 16β-methyl-16,17-oxido-11α-hydroxypregna-1,4,6-triene-3,20-dione (IV), and the intermediate product as 16β-methyl-16,17-oxido-11α-hydroxypregna-4,6-diene-3,20-dione (III).