The microsomal demethylation of N,N-dimethylbenzamides. Substituent and kinetic deuterium isotope effects.

The metabolism of N,N-dimethylbenzamides by phenobarbital-induced rat liver microsomes results in the formation of N-methylbenzamides and formaldehyde. The reaction proceeds via the formation of an intermediate N-hydroxymethyl-N-methylbenzamide, which, for the microsomal oxidation of N,N-dimethylbenzamide, was isolated and characterized. Confirmation of the N-hydroxymethyl-N-methylbenzamide was obtained by its independent synthesis from N-methylbenzamide and formaldehyde. The intermolecular kinetic deuterium isotope effects for the reaction are 0.9 (+/- 0.1) for Vmax and 1.4 (+/- 0.1) for Vmax/Km. The intramolecular kinetic deuterium isotope effect, determined from the relative amounts of N-methylbenzamide and N-trideuteriomethylbenzamide formed in the microsomal demethylation of N-trideuteriomethyl-N-methylbenzamide, is 6.0 +/- 0.3. There is no correlation of Vmax or Vmax/Km with the substituent in the aromatic ring, nor with the calculated ionization potentials of the benzamides. The results are interpreted in terms of a mechanism in which the benzamide undergoes direct hydrogen atom abstraction to form a carbon centred radical. This carbon centred radical subsequently forms an N-hydroxymethyl-N-methylbenzamide that decomposes to formaldehyde and an N-methylbenzamide. Semi-empirical AM1 self consistent field molecular orbital calculations identify that loss of a hydrogen atom from the E-methyl group is thermodynamically more favourable than from the Z-methyl group by ca. 5 kJ/mol.     

Secondary deuterium isotope effects on olefin epoxidation by cytochrome P-450

Secondary deuterium isotope effects have been determined for the epoxidation of p-phenylstyrene (1a) and p-methylstyrene (1b) by cytochrome P-450 of rat liver microsomes. With both substrates there is an inverse isotope effect of 7 per cent/deuterium (i.e. $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{<mtext>D</mtext>}}\text{= 0.93}$) at C_α of the olefin, but no isotope effect is observed at C_β. The epoxidation of (1a) by m-chloroperbenzoic acid has previously been shown to be accompanied by an inverse secondary isotope effect of 9 if per cent/deuterium at C_β, with no detectable isotope effect at C_α. Thus in both the enzymatic (P-450) and non-enzymatic (peracid) epoxidation of styrene derivatives, the oxygen atom is transferred to the vinyl group in an asymmetric non-concerted fashion. However, the fact that the isotope effects for these two systems are reversed, together with previous comparisons of substituent effects on the two reactions, suggests that there is little mechanistic similarity between cytochrome P-450 enzymes and organic peracids as chemical models for these enzymes.     

Procarbazine spermatogenesis toxicity: deuterium isotope effects point to regioselective metabolism in mice

Procarbazine was shown to decrease spermatogenesis in male mice in a dose-dependent manner. Significant decreases (44% of controls) in spermatogenesis were observed when a dose of 400 mg/kg was administered 18 days prior to determination of sperm count. Procarbazine caused no significant acute spermatocidal activity in vivo. Procarbazine-associated decreases in spermatogenesis were thus used as an index of toxicity to developing spermatid cells. Procarbazine analogs were synthesized that had deuterium substituted for hydrogen at the benzylic position, N-isopropyl-alpha-(2-methylhydrazino)-p-[alpha, alpha-2H2]toluamide (d2-procarbazine), or at the methyl position, N-isopropyl-alpha-(2-[alpha, alpha, alpha-2H3]methylhydrazino)-p-toluamide (d3-procarbazine). Spermatogenesis decreases caused by d3-procarbazine were essentially the same as with procarbazine in mice (66% of controls at a dose of 200 mg/kg), but d2-procarbazine was nontoxic to developing sperm cells (99% of control at a dose of 200 mg/kg). The decrease in toxicity caused by deuterium substitution at the benzylic position, coupled with the absence of an effect with the methyl-labeled analog, indicate the requirement for regioselective oxidative metabolism of procarbazine at the benzylic position prior to the toxic event.     

The use of deuterium isotope effects to probe the active site properties, mechanism of cytochrome P450-catalyzed reactions, and mechanisms of metabolically dependent toxicity.

Critical elements from studies that have led to our current understanding of the factors that cause the observed primary deuterium isotope effect, (kH/kD)obs, of most enzymatically mediated reactions to be much smaller than the "true" or intrinsic primary deuterium isotope effect, kH/kD, for the reaction are presented. This new understanding has provided a unique and powerful tool for probing the catalytic and active site properties of enzymes, particularly the cytochromes P450 (P450). Examples are presented that illustrate how the technique has been used to determine kH/kD, and properties such as the catalytic nature of the reactive oxenoid intermediate, prochiral selectivity, the chemical and enzymatic mechanisms of cytochrome P450-catalyzed reactions, and the relative active site size of different P450 isoforms. Examples are also presented of how deuterium isotope effects have been used to probe mechanisms of the formation of reactive metabolites that can cause toxic effects.     

Determination of the mechanism of demethylenation of (methylenedioxy)phenyl compounds by cytochrome P450 using deuterium isotope effects.

The mechanism of demethylenation of (methylenedioxy)benzene (MDB), (methylenedioxy)amphetamine (MDA), and (methylenedioxy)methamphetamine (MDMA) by purified rabbit liver cytochrome P450IIB4 has been investigated by using deuterium isotope effects. A comparison of the magnitude and direction of the observed kinetic isotope effects indicates that the three compounds are demethylenated by different mechanisms. The different mechanisms of demethylenation have been proposed on the basis of comparisons of the observed biochemical isotope effects with the isotope effects from purely chemical systems.     

蒿甲醚经大鼠肝微粒体酶脱甲基代谢和氘代同位素效应

经苯巴比妥钠诱导前后的大鼠肝微粒体酶对蒿甲醚脱甲基代谢的Km分别为0.12和0.19mmol/L,Vm分别为0.42和3.97nmol·min~(-1)·mg~(-1)蒿甲醚经诱导酶代谢,证明有氘代同位素效应(DIE)。测定甲醛生成DIE值为2.37±0.08;测定蒿甲醚消失DIE值为1.66±0.08。估算氘代和非氘代蒿甲醚脱甲基率分别为37和53％。提示蒿甲醚的代谢与NADPH-细胞色素P450系统密切相关,O-脱甲基是其主要代谢途径。     

Physicochemical properties of beta-lactam antibacterials: deuterium solvent isotope effect on penicillin G degradation rate.

To obtain kinetic evidence on the degradation mechanism of penicillin in aqueous solution, degradation rates of penicillin G in water and deuterium oxide were measured in the pH (pD) range of 4-10. The solvent isotope effect (kH2O/kD2O) of 1.53 below pH (pD) 6 supports the mechanism of water-catalyzed rearrangement of undissociated penicillin G to benzylpenicillenic acid. The spontaneous degradation at neutral pH (pD) and the hydroxide-ion-catalyzed degradation in the alkaline pH (pD) range progress with a deuterium solvent isotope effect (kH2O/kD2O) of 4.5 and 0.59, respectively. This finding indicates the mechanisms of general base-catalyzed hydrolysis by water in the neutral pH range and of nucleophilic attack of the hydroxide ion on the beta-lactam in the alkaline pH range. No significant side-chain dependency was observed in the reaction of penicillins with bases. The solvent isotope studies led to the conclusion that penicillin degradation is catalyzed by a series of bases via general base-catalyzed and nucleophilic mechanisms, depending on their basicity.     

Toxic reactions and side effects of glucocorticoids in man.

The toxic reactions and side effects of glucocorticoids in humans are discussed. The effects reviewed include metabolic (muscle and skin, carbohydrates and lipids), cardiovascular, cellular, immunologic, skeletal and growth, gastrointestinal, nervous system, ocular, and hypothalmic-pituitary-adrenal.     

Rate-limiting steps in cytochrome P-450-catalysed reactions: Studies on isotope effects in the O-de-ethylation of 7-ethoxycoumarin

1. Primary deuterium isotope effects ranging from 2 to 6 were measured for the O-de-ethylation of 7-ethoxycoumarin catalysed by microsomal and purified cytochrome P-450 isozymes.

2. Interpretation of the observed deuterium isotope effect in terms of the contribution of the C-H bond-cleavage step to the overall rate requires the determination of the intrinsic isotope effect.

3. Evidence is presented that at least one irreversible step occurs before C-H bond cleavage which kinetically divides the cytochrome P-450 reaction cycle into two portions. Intermolecular primary isotope effects are shown to provide kinetic information only about steps following the irreversible step. Consequently, the isotope effect may be a measure of the rate limitation imposed by the C-H bond-cleavage step in the sequence of events following the irreversible step but be totally independent of other slow steps preceding the irreversible step. These results indicate that multiple rate-limiting steps may exist in cytochrome P-450-catalysed reactions.