Generation and fate of enamines in the microsomal metabolism of cyclic tertiary amines

Sayre, Lawrence M.; Engelhart, David A.; Venkataraman, B.; Babu, M. K. Manoj; McCoy, G. David

doi:10.1016/0006-291x(91)91724-q

Cited by 11 publications

(13 citation statements)

References 23 publications

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“…In this particular case, the main reaction products were two α-aminonitriles (49 and 50 in Scheme 5), counting for about 86% of the reacted 1. They clearly resulted from the iminium cations 44 and 45, respectively, by cyano trapping [7,8,[27][28][29]. Excepting 4, the other reaction products (i.e., 2+7 and 30+BzH) were oxygen-containing compounds, clearly originating from the same iminium cations, escaped from the cyanide anion trapping (i.e., captured by water).…”

Section: Scheme 4 Oxidation By Ruo 4 Of Piperazine Derivative 30 ([Oxmentioning

confidence: 98%

RuO4-mediated oxidation of N-benzylated tertiary amines. 3. Behavior of 1,4-dibenzylpiperazine and its oxygenated derivatives

Petride¹,

Drăghici²,

Florea³

et al. 2006

Open Chemistry

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1,4-Dibenzylpiperazine (1), -2-piperazinone (7), -2,6-piperazinedione (9), and 1-benzoyl-4-benzylpiperazine (30) were oxidized by RuO 4 (generated in situ) by attack at their endocyclic and exocyclic (i.e., benzylic) aminic N -α-C-H bonds to afford various oxygenated derivatives, including acyclic diformamides, benzaldehyde, and benzoic acid. The reaction outcome was complicated by (i) the hydrolysis of diformamides, occurred during the work-up, and (ii) the reaction of benzaldehyde with the hydrolysis-derived amines giving imidazolidines and/or Schiff bases. Benzoic acid resulted from benzaldehyde only. Compounds 7, 30, and 1-benzylpiperazine, but not 9, were transiently formed during the oxidation of 1. In the same reaction conditions, 1,4-dibenzyl-2,3-(or 2,5)-piperazinedione, 1,4-dibenzyl-2,3,6-piperazinetrione, 4-benzoyl-1-benzyl-2-piperazinone, and 1,4-dibenzoylpiperazine were inert. The proposed oxidation mechanism involves the formation of endocyclic and exocyclic iminium cations, as well as of cyclic enamines. The latter intermediates probably result by base-induced deprotonation of the iminium cations, provided an N + −β-proton is available. In the case of 1, the cations were trapped with NaCN as the corresponding α-aminonitriles. The statistically corrected regioselectivity (endocyclic/exocyclic) of the RuO 4 -induced oxidation reaction of 1, 7, and 30 was 1.2-1.3.

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Section: Scheme 4 Oxidation By Ruo 4 Of Piperazine Derivative 30 ([Oxmentioning

confidence: 98%

RuO4-mediated oxidation of N-benzylated tertiary amines. 3. Behavior of 1,4-dibenzylpiperazine and its oxygenated derivatives

Petride¹,

Drăghici²,

Florea³

et al. 2006

Open Chemistry

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“…This frequently results in poor pharmacokinetic properties for the new drug candidates. The N-substituted piperidines are also prone to metabolic activation via the formation of reactive iminium ions that may covalently modify proteins and other cellular nucleophiles and potentially result in toxicity (Sayre et al, 1991;Gorrod and Aislaitner, 1994;Castagnoli et al, 1997). Iminium ion formation also presumably involves oxidation at the piperidine ring ␣-carbons (Castagnoli et al, 1997).…”

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confidence: 99%

A Novel P450-Catalyzed Transformation of the 2,2,6,6-Tetramethyl Piperidine Moiety to a 2,2-Dimethyl Pyrrolidine in Human Liver Microsomes: Characterization by High Resolution Quadrupole-Time-of-Flight Mass Spectrometry and ¹H-NMR

Yin

Doss²,

Stearns³

et al. 2003

Drug Metab Dispos

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ABSTRACT:We describe herein a novel metabolic fate of the 2,2,6,6-tetramethyl-piperidine (2,2,6,6-TMPi) moiety to a ring-contracted 2,2-dimethyl pyrrolidine (2,2-DMPy) in human liver microsomal incubations. The existence of this pathway was demonstrated for three compounds (I-III) of varied structures suggesting that this may be a general biotransformation reaction for the 2,2,6,6-TMPi moiety. The 2,2-DMPy metabolites formed in incubations of the three compounds with human liver microsomes were characterized by online high performance liquid chromatography coupled to a high resolution hybrid quadrupole-time-of-flight mass spectrometer. Suggested elemental composition obtained from accurate mass measurements of the molecular ions and fragment ions of the metabolites clearly indicated the loss of a mass equivalent to C 3 H 6 from the parent 2,2,6,6-TMPi functionality. Additional accurate tandem mass spectrometry data indicated that one of the original two gem-dimethyl groups was intact in the metabolite structure. Proof of a ring-contracted 2,2-DMPy structure was obtained using 1 H-NMR experiments on a metabolite purified from liver microsomal incubations, which showed only two geminal methyl groups, instead of four in the parent compound. Two-dimensional correlation spectroscopy and decoupling experiments established aliphatic protons arranged in a pyrrolidine ring pattern. The fact that the formation of 2,2-DMPy metabolites in human liver microsomes was NADPH-dependent suggested that this novel metabolic reaction was catalyzed by the cytochrome P450 (P450) enzyme(s). Immunoinhibition studies in human liver microsomal incubations using anti-P450 monoclonal antibodies and experiments with insect cell microsomes containing individually expressed recombinant human P450 isozymes indicated that multiple P450 isozymes were capable of catalyzing this novel metabolic transformation.

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“…This hypothesis was supported by the formation of PCP iminium-cyanide adducts and the observation that the presence of cyanide in microsomal incubations protected against the formation of covalent adducts with proteins (Ward et al, 1982b;Hoag et al, 1984). However, subsequent studies demonstrated that the P450 inactivation by the PCP iminium ion (PCP-Im) required the presence of NADPH (Hoag et al, 1984;Osawa and Coon, 1989;Sayre et al, 1991), which inferred that metabolism of PCP beyond the iminium species was required for P450 inactivation.Later, the PCP mechanism-based inactivation of P450 2B6, using purified, recombinant human P450 enzyme, was investigated in great detail (Jushchyshyn et al, 2003). Through a series of experiments, it was shown that P450 2B6 was inactivated via covalent modification of the P450 2B6 apoprotein.…”

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confidence: 99%

Mechanism of Inactivation of Human Cytochrome P450 2B6 by Phencyclidine

Jushchyshyn¹,

Wahlstrom

Hollenberg³

et al. 2006

Drug Metab Dispos

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ABSTRACT:The mechanism behind the observed inactivation of human P450 2B6 by phencyclidine (PCP) has been evaluated over the past 2 decades. The scope of the current investigation was to contribute to the fundamental knowledge of PCP oxidation and perhaps the mechanism behind P450 inactivation. To study the chemistry of PCP oxidation, we subjected PCP to the Fenton reagent. Under Fenton chemistry conditions, oxidation on all three PCP rings was observed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). When PCP was incubated with the Fenton system in the presence of glutathione (GSH), three GSH-PCP conjugates were identified. Subsequent LC-MS/MS analysis of these conjugates revealed two species that had GSH attached to the cyclohexane ring of PCP and a third conjugate in which GSH was adducted to the piperidine ring. When PCP was incubated across a panel of P450 enzymes, several enzymes, including P450s 2D6 and 3A4, were able to catalyze the formation of the PCP iminium ion, whereas P450s 2B6 and 2C19 were exclusively able to hydroxylate secondary carbons on the cyclohexane ring of PCP. Subsequent mechanistic experiments revealed that only P450s 2B6 and 2C19 demonstrated loss of catalytic activity after preincubation with 10 M PCP. Finally, investigation of P450 2B6 inactivation using structural analogs of PCP revealed that blocking the para-carbon atom on the cyclohexane ring of PCP from oxidation protected the P450 2B6 from inactivation, which suggests that a reactive intermediate generated during the hydroxylation of the cyclohexane ring may be linked to the mechanism of inactivation of P450 2B6 by PCP.The biotransformation of phencyclidine, PCP (Fig. 1), by liver proteins was shown to result in the formation of reactive metabolites leading to P450 inactivation and the formation of covalent adducts with hepatic macromolecules more than 20 years ago (Castagnoli et al., 1997). Originally, covalent binding and enzyme inactivation was thought to occur via alkylation of protein-based nucleophiles by an iminium ion metabolite, M4 (Fig. 1), that arose from the P450-dependent two-electron ␣-carbon oxidation of PCP (Sayre et al., 1997). This hypothesis was supported by the formation of PCP iminium-cyanide adducts and the observation that the presence of cyanide in microsomal incubations protected against the formation of covalent adducts with proteins (Ward et al., 1982b;Hoag et al., 1984). However, subsequent studies demonstrated that the P450 inactivation by the PCP iminium ion (PCP-Im) required the presence of NADPH (Hoag et al., 1984;Osawa and Coon, 1989;Sayre et al., 1991), which inferred that metabolism of PCP beyond the iminium species was required for P450 inactivation.Later, the PCP mechanism-based inactivation of P450 2B6, using purified, recombinant human P450 enzyme, was investigated in great detail (Jushchyshyn et al., 2003). Through a series of experiments, it was shown that P450 2B6 was inactivated via covalent modification of the P450 2B6 apoprotein. Moreover, inclusion of cyanide in the pu...

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Generation and fate of enamines in the microsomal metabolism of cyclic tertiary amines

Cited by 11 publications

References 23 publications

RuO4-mediated oxidation of N-benzylated tertiary amines. 3. Behavior of 1,4-dibenzylpiperazine and its oxygenated derivatives

RuO4-mediated oxidation of N-benzylated tertiary amines. 3. Behavior of 1,4-dibenzylpiperazine and its oxygenated derivatives

A Novel P450-Catalyzed Transformation of the 2,2,6,6-Tetramethyl Piperidine Moiety to a 2,2-Dimethyl Pyrrolidine in Human Liver Microsomes: Characterization by High Resolution Quadrupole-Time-of-Flight Mass Spectrometry and ¹H-NMR

Mechanism of Inactivation of Human Cytochrome P450 2B6 by Phencyclidine

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Generation and fate of enamines in the microsomal metabolism of cyclic tertiary amines

Cited by 11 publications

References 23 publications

RuO4-mediated oxidation of N-benzylated tertiary amines. 3. Behavior of 1,4-dibenzylpiperazine and its oxygenated derivatives

RuO4-mediated oxidation of N-benzylated tertiary amines. 3. Behavior of 1,4-dibenzylpiperazine and its oxygenated derivatives

A Novel P450-Catalyzed Transformation of the 2,2,6,6-Tetramethyl Piperidine Moiety to a 2,2-Dimethyl Pyrrolidine in Human Liver Microsomes: Characterization by High Resolution Quadrupole-Time-of-Flight Mass Spectrometry and 1H-NMR

Mechanism of Inactivation of Human Cytochrome P450 2B6 by Phencyclidine

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A Novel P450-Catalyzed Transformation of the 2,2,6,6-Tetramethyl Piperidine Moiety to a 2,2-Dimethyl Pyrrolidine in Human Liver Microsomes: Characterization by High Resolution Quadrupole-Time-of-Flight Mass Spectrometry and ¹H-NMR