Rat liver microsomal enzyme catalyzed oxidation of 4-phenyl-trans-1-(2-phenylcyclopropyl)-1,2,3,6-tetrahydropyridine.

As part of our ongoing studies to characterize the catalytic pathway(s) for the monoamine oxidase and cytochrome P450 catalyzed oxidations of 1,4-disubstituted 1,2,3,6-tetrahydropyridinyl derivatives, we have examined the metabolic fate of 4-phenyl-trans-1-(2-phenylcyclopropyl)-1,2,3,6-tetrahydropyridine in NADPH supplemented rat liver microsomes. Three metabolic pathways have been identified: (1) allylic ring alpha-carbon oxidation to yield the dihydropyridinium species, (2) nitrogen oxidation to yield the N-oxide and (3) N-dealkylation to yield 4-phenyl-1,2,3,6-tetrahydropyridine and cinnamaldehyde. A possible mechanism to account for the formation of cinnamaldehye involves an initial single electron transfer from the nitrogen lone pair to the iron oxo system Fe(+3)(O) to form the corresponding cyclopropylaminyl radical cation that will be processed further to the final products. The reaction pathway leading to the dihydropyridinium metabolite may also proceed via the same radical cation intermediate but direct experimental evidence to this effect remains to be obtained.

Rat liver microsomal enzyme catalyzed oxidation of 1-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine.

NADPH supplemented rat liver microsomal enzyme preparations catalyze the conversion of 1-cyclopropyl4-phenyl-1,2,3,6-tetrahydropyridine to the p-hydroxyphenyl (low yield), descyclopropyl (high yield) and 2,3-dihydropyridinium and, subsequently, pyridinium (intermediary yield) metabolites. When the methine proton of the cyclopropyl group was replaced with a deuteron, a normal deuterium isotope effect (1.4) was observed on the formation of the decyclopropylated metabolite and an inverse isotope effect (0.6) on the dihydropyridinium metabolite. A larger deuterium isotope effect (3.6) was observed on the ring alpha-carbon oxidation pathway with the 2,2,6,6-d4 analogue as substrate. These results and the observation that the ratios of the rates of these two alpha-carbon oxidation pathways are independent of initial substrate concentrations suggest that both pathways are catalyzed by the same active site of one form of P450. These transformations are discussed in terms of metabolic pathways that have been proposed for the cytochrome P450 catalyzed alpha-carbon oxidation of amines.

Deuterium isotope effect studies on the MAO-B catalyzed oxidation of 4-benzyl-1-cyclopropyl-1,2,3,6-tetrahydropyridine.

Previous studies have established that 1-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine is an efficient time- and concentration-dependent inhibitor of the flavin-containing enzyme monoamine oxidase B (MAO-B). This behavior is consistent with a proposed mechanism-based inactivation pathway which proceeds via an initial single electron transfer step to generate an unstable cyclopropylaminyl radical cation intermediate that alkylates an active site functionality via the ring opened primary carbon centered radical. More recently we have found that, in addition to being an inhibitor, the corresponding 1-cyclopropyl-4-benzyl-1,2,3,6-tetrahydropyridine species is an excellent MAO-B substrate, behavior which may not be consistent with the obligatory formation of a cyclopropylaminyl radical cation intermediate. In an attempt to gain further insight into the mechanism associated with the MAO catalyzed oxidation of 1,4-disubstituted tetrahydropyridines, we have undertaken deuterium isotope effect studies on the substrate and inhibitor properties of this 4-benzyl-1-cyclopropyltetrahydropyridine derivative. A normal isotope effect was observed on kcat/KM. Although the good substrate properties of this compound prevented an accurate estimate of k(inact) and K1, we did observe a very modest inverse isotope effect on the rate of inactivation of 0.1 microM MAO-B by 500 microM inactivator. The results are discussed in terms of possible mechanisms for the MAO-B catalyzed oxidation of 1,4-disubstituted 1,2,3,6-tetrahydropyridines.

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.

Mechanistic studies on the monoamine oxidase B catalyzed oxidation of 1,4-disubstituted tetrahydropyridines.

Previous studies have established that 1-cyclopropyl-4-phenyl-1,2,3,6- tetrahydropyridine (6) is an efficient time and concentration dependent inhibitor of the flavin containing enzyme monoamine oxidase B (MAO-B). This behavior is consistent with a proposed mechanism based inactivation pathway initiated by transfer of one of the nitrogen nonbonding pairs of electrons to the oxidized flavin cofactor to generate an amine radical cation intermediate. Subsequent opening of the strained cyclopropylamine ring is thought to lead to a primary carbon centered radical that inactivates the enzyme by covalent modification of the flavin or an essential active site functionality. We now have examined the MAO-B inactivator and substrate properties of 4-benzyl-1-cyclopropyl-1,2,3,6-tetrahydropyridine (11). This compound also is a time and concentration dependent inhibitor of MAO-B. Unexpectedly, however, compound 11 proved to be an excellent MAO-B substrate. These results are discussed in terms of possible catalytic pathways for the MAO-B catalyzed oxidation of 1,4-disubstituted-1,2,3,6- tetrahydropyridines.

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.