Probing the mechanism of bioactivation of MPTP type analogs by monoamine oxidase B: structure-activity studies on substituted 4-phenoxy-, 4-phenyl-, and 4-thiophenoxy-1-cyclopropyl-1,2,3,6-tetrahydropyridines.

Previous studies have shown that 4-benzyl-1-cyclopropyl-1,2,3,6-tetrahydropyridine is an excellent monoamine oxidase B (MAO-B) substrate (kappa cat/KM = 1538 min-1 mM-1) although the corresponding 4-phenyl analog displays MAO-B inactivating properties only. This behavior led us to speculate that the pathway for the MAO-B catalyzed oxidation of these tetrahydropyridines may not necessarily proceed via an initial single electron transfer step as proposed by others but rather through an initial alpha-carbon hydrogen atom abstraction step. In the present studies we have examined the interactions of various 4-phenoxy-, 4-phenyl-, and 4-thiophenoxy-1-cyclopropyl-1,2,3,6-tetrahydropyridine derivatives, some of which bear substituents on the phenyl ring. The 4-thiophenoxy- and all of the 4-phenoxytetrahydropyridine derivatives proved to be substrates but not inactivators of MAO-B, while several of the 4-phenyltetrahydropyridine derivatives were inactivators but not substrates. A case of particular interest was 1-cyclopropyl-4-(2-methylphenyl)-1,2,3,6-tetrahydropyridine, which displayed only substrate properties. The results are discussed in terms of two catalytic pathways, one of which involves partitioning of the proposed cyclopropylaminyl radical cation intermediate between cyclopropyl ring opening and proton loss while the second involves partitioning of the parent amine between an initial single electron transfer step, leading to cyclopropylaminyl radical cation formation and enzyme inactivation, and an initial alpha-carbon hydrogen atom abstraction step, leading to an allylic radical and dihydropyridinium product formation.

Effect of phenobarbital pretreatment on the plasma and urinary levels of (-)-alpha-acetylmethadol and its metabolites.

The effect of phenobarbital (PB), an inducer of the hepatic microsomal enzyme system, on the plasma levels and urinary elimination of (-)-alpha-acetylmethadol 1 and its metabolites have been examined in the rat. [3H]1 was administered to saline control and PB-pretreated rats at doses of 5 mg/kg ip (55 muCi/kg). The concentration of 1 and its metabolites noracetylmethadol 2, dinoracetylmethadol 3, methadol 4, normethadol 5, and N-acetylnormethadol 6 were quantitated in plasma and urine over 48 h by TLC and liquid scintillation counting. PB pretreatment significantly decreased the plasma total radioactivity and the levels of 1 and its five metabolites over the 48-h period investigated. Urinary total radioactivity and elimination of 1 and its five metabolites were also reduced in PB-pretreated rats. The results indicated that PB pretreatment markedly affects the in vivo transformation and elimination of 1 and its metabolites. The decrease in the levels observed for 1 and its metabolites in the plasma and urine can be due either to an increase in the metabolism of 1 via a different pathway than the formation of the biologically active metabolites 2, 3, 4, and 5, or it may be that PB is enhancing the further metabolism of these compounds to more polar water-soluble products which are mainly excreted through the bile.

Relationship between azo dye structure and rat hepatic azoreductase activity.

The rate of reduction was determined for a variety of azo dyes using the rat hepatic azoreductase enzyme system. In decreasing order, the rates of reduction for the azo dyes expressed as nmol of arylamine product formed/min/0.25 g of liver were amaranth (33.2), azosulfamide (32.5), orange G (12.4), 1,2-dimethyl-4-p-(carboxyphenylazo)-5-hydroxybenzene (CPA) (9.27), brilliant crystal scarlet (8.00), sulfachrysoidine (7.27), and Sudan I (1.03). A comparison of the partition coefficient with its rate of reduction indicated that the water-soluble azo dyes were reduced more rapidly than the lipid-soluble ones. Furthermore, higher rates of reduction were observed for those dyes containing electron-withdrawing groups on the aromatic rings.

Analysis of N-n-propylnorapomorphine in plasma and tissue by capillary gas chromatography--electron-capture detection.

Capillary gas chromatography combined with electron-capture detection (GC-ECD) was applied to the detection and quantitation of N-n-propylnorapomorphine (NPA) and related compounds in serum and tissue using trifluoroacetyl (TFA) derivatives. The detection limits for NPA using GC-ECD of TFA derivative extend into the subpicogram level, but quantitation in serum was limited to levels of 100 ng/ml due to matrix interferences. The method was applied to the analysis of NPA in rat serum after administration of a moderate dose of the drug and was applied to the detection of NPA in rat brain after the peripheral administration of (-)10,11-methylenedioxy-N-n-propylnoraporphine (MDO-NPA). These results support previous proposals that MDO-NPA is a prodrug of NPA, which acts at cerebral dopamine-receptors.

Structural considerations in the metabolism of nitriles to cyanide in vivo.

In order to investigate structure-activity relationships that influence metabolism of nitriles to CN-, thiocyanate was measured, as an index of CN- release, in urine of rats given equimolar doses of nitriles. Significantly more SCN- was excreted after po than after ip administration of saturated (C2-C5) nitriles, but SCN- excretion was the same after both routes for n-hexanenitrile. Among saturated nitriles, SCN- excretion was maximal for the C3 and C4 compounds, propionitrile, n-butyronitrile, and isobutyronitrile, after both po and ip administration. SCN- excretion was not elevated after administration of the tertiary nitrile trimethylacetonitrile. Administration (po) of the unsaturated nitriles acrylonitrile, crotonitrile, and 3-butenenitrile yielded 37%, 5.6%, and 29% of the dose as SCN-, whereas after ip injection 4.5%, 4.6%, and 18% of the doses were excreted as SCN-, respectively. After iv injection of acrylonitrile, urinary SCN- content was not elevated, whereas 45% of an iv dose of the saturated analog propionitrile was excreted as SCN-. These results suggest that length of the carbon chain, presence of substituents at the alpha-carbon, position of double bonds, and, for some compounds, route of administration, are important factors influencing the release of CN- from nitriles.

Studies on the metabolism of procarbazine by mass spectrometry.

The mass spectrometric properties of procarbazine and some of its breakdown products were examined using isotopic labeling and high resolution mass spectrometry. A specific intramolecular methyl group transfer, induced by electron impact, was used diagnostically to distinguish between two isomeric derivatives. The mass spectrometric information was used for identification of the in vivo metabolites of procarbazine in rat. Minimum sample manipulation was used due to the inherent instability of the suspected metabolites. The azo compound N-isopropyl- alpha -(2-methylazo)-p-toluamide was identified as the principal circulating metabolite of procarbazine.

Isolation of melanin from human plasma lipofuscin.

Human plasma lipofuscin and its melanin component were isolated and quantified. Electron paramagnetic resonance, infrared, ultraviolet and visible spectra of this melanin exhibited absorption characteristics very similar to those of known melanins. The human plasma lipofuscin contained approximately 85% protein, 3% melanin, 0.4% lipid and 0.25% mucoprotein constituents and emitted yellow-green fluorescence in 366-nm light. The ethanol-ether lipid extract obtained after acid hydrolysis from the lipid-melanin fraction of this lipofuscin was also found to fluoresce in yellow-green color in 366-nm light and produced similar fluorescence excitation and emission spectra as those of the human plasma lipofuscin in water solution. The isolated melanin component was not fluorescent.

Studies on rheomelanins. VI. The apparent lipofuscin characteristics of rheomelanins.

The positive histochemical tests obtained on rheomelanins indicate a relationship with tissue lipofuscins, which are also melanins in part. In addition chemical analysis indicates that the rheomelanins contain protein and lipid just as lipofuscins do. Fluorescence exhibited by the rheomelanins also seems to ally them to the lipofuscins.

Conformationally defined aromatic amino acids. Synthesis and stereochemistry of 2-endo- and 2-exo-amino-1,2,3,4-tetrahydro-1,4-ethanonaphthalene-2-carboxylic acids (2-endo- and 2-exo-aminobenzobicycle[2.2.2]octene-2-carboxylic acids).

The synthesis of the title compounds 1a and 1b has been accomplished in good yield by conversion of ketone 3 to the corresponding hydantoins 4a and 4b via a Bucherer-Bergs reaction, followed by barium hydroxide hydrolysis. The stereochemical assignments of the intermediate hydantoins 4a and 4b and the ethyl ester hydrochlorides 5a and 5b were determined by H NMR analysis. Attempts toward the synthesis of 2-amino-1,4-dihydro-1,4-ethanonaphthalene-2-carboxylic acid isomers 2a and 2b utilizing the pathway discussed for 1a and 1b led only to products arising from a retro-Diels-Alder reaction. Preliminary screening of 1a and 1b as inhibitors of phenylalanine hydroxylase (PH) and phenylalanine decarboxylase (PAD) is also discussed. The use of the benzobicyclo[2.2.2]octene nucleus for the construction of conformationally defined analogues of important medicinal agents is rationalized, and the title compounds are compared to several other conformationally defined systems. The title compounds represent conformationally defined models of the lower energy conformations of alpha-methylphenylalanine.

Metabolism and disposition of l-alpha-acetylmethadol in the rat.

The metabolism and disposition of the long-acting narcotic analygesic l-alpha-acetylmethadol (LAAM) were studied in the rat. 3H-LAAM was administered to male and female rats at doses of 5 mg/kg po and iv, and 10 mg/kg po. LAAM was rapidly absorbed and extensively metabolized. Five metabolites-noracetylmethadol, dinoracetylmethadol, methadol, normethadol, and N-acetylnormethadol-were identified in plasma and urine. Feces were the major route of elimination for the parent drug and metabolites. Less than 20% of the administered dose was excreted in the urine and, of this, greater than 90% was in the form of conjugates or polar metabolites. There is an apparent sex-related difference in LAAM disposition in the rat. LAAM and metabolites tended to persist in higher levels in female rats as compared with male rats. Similarly, male rats tended to excrete the drug at a faster rate than did females.

Extraction method and thin-layer chromatographic system for the determination of alpha-l-acetylmethadol and metabolites in biological fluids.

An extraction method and thin-layer chromatographic (TLC) system for the determination of alpha-l-acetylmethadol and its known metabolites (methadol, noracetylmethadol, dinoracetylmethadol, normethadol, 6-acetamide-4,4-diphenyl-3-heptanol, and N-methyl-6-acetamido-4,4-diphenyl-3-heptanol) are described. The parent drug and metabolites are extracted from biological fluids with ethyl acetate and separated by TLC using silica gel plates and a developing system of ethyl acetate-methanol-water-ammonia (85:10:1:1). This system may be used to quantitatively determine levels of radiolabeled drug and metabolites by scraping the TLC plates into 3-mm zonal fractions and measuring the amount of radioactivity by scintillation counting. A representative radiochromatogram obtained from an extract of monkey urine is shown.