Deuterium-isotope effect in the biotransformation of 4-ethynylbiphenyls to 4-biphenylylacetic acids by rat hepatic microsomes

McMahon, Robert E.; Turner, Judy C.; Whitaker, G. W.; Sullivan, Hugh R.

doi:10.1016/0006-291x(81)91795-2

Cited by 13 publications

(4 citation statements)

References 5 publications

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“…Also, the substrateinduced inactivation of aromatase by the formation of oxirene intermediates from acetylinic steroids had been proposed previously (19). The oxidation of 4-ethynylbiphenyl to the carboxylic acid was postulated to involve an oxirene, since the labeled alkyne gave an acid product with complete retention of deuterium (8,18,20). The mechanism for the retention of deuterium of terminal acetylene was believed to be due to the formation of a ketene with the migration of deuterium.…”

Section: Discussionmentioning

confidence: 93%

Characterization of Novel Glutathione Adducts of a Non-Nucleoside Reverse Transcriptase Inhibitor, (S)-6-Chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3,4-dihydro-2(1H)-quinazolinone (DPC 961), in Rats. Possible Formation of an Oxirene Metabolic Intermediate from a Disubstituted Alkyne

Mutlib

Chen

Shockcor

et al. 2000

Chem. Res. Toxicol.

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The postulated formation of oxirene-derived metabolites from rats treated with a disubstituted alkyne, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), is described. The reactivity of this postulated oxirene intermediate led to the formation of novel glutathione adducts whose structures were confirmed by LC/MS and by two-dimensional NMR experiments. These metabolites were either excreted in rat bile or degraded to mercapturic acid conjugates and eliminated in urine. To demonstrate the oxidation of the triple bond, an analogue of DPC 961 was synthesized, whereby the two carbons of the alkyne moiety were replaced with (13)C stable isotope labels. Rats were orally administered [(13)C]DPC 961 and glutathione adducts isolated from bile. The presence of an oxygen atom on one of the (13)C labels of the alkyne was demonstrated unequivocally by NMR experiments. Administration of (14)C-labeled DPC 961 showed that biliary elimination was the major route of excretion with the 8-OH glucuronide conjugate (M1) accounting for greater than 90% of the eliminated radioactivity. On the basis of radiochemical profiling, the glutathione-derived metabolites were minor in comparison to the glucuronide conjugate. Studies with cDNA-expressed rat enzymes, polyclonal antibodies, and chemical inhibitors pointed to the involvement of P450 3A1 and P450 1A2 in the formation of the postulated oxirene intermediate. The proposed mechanism shown in Scheme 1 begins with P450-catalyzed formation of an oxirene, rearrangement to a reactive cyclobutenyl ketone, and a 1,4-Michael addition with endogenous glutathione to produce two isomeric adducts, GS-1 and GS-2. The glutathione adducts were subsequently catabolized via the mercapturic acid pathway to cysteinylglycine, cysteine, and N-acetylcysteine adducts. The transient existence of the alpha,beta-unsaturated cyclobutenyl ketone was demonstrated by incubating the glutathione adduct in the presence of N-acetylcysteine and monitoring the formation of N-acetylcysteine adducts by LC/MS. Epimerization of GS-1 to GS-2 was also observed when N-acetylcysteine was omitted from the incubation.

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Section: Discussionmentioning

confidence: 93%

Characterization of Novel Glutathione Adducts of a Non-Nucleoside Reverse Transcriptase Inhibitor, (S)-6-Chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3,4-dihydro-2(1H)-quinazolinone (DPC 961), in Rats. Possible Formation of an Oxirene Metabolic Intermediate from a Disubstituted Alkyne

Mutlib

Chen

Shockcor

et al. 2000

Chem. Res. Toxicol.

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“…The validity of this proposal is discussed below. An alternative mechanism envisions insertion of oxygen into the acetylenic C-H bond, followed by rearrangement to the ketene (Figure 3, path b) (McMahon et al 1981). However, this latter mechanism is highly disfavored by the C-H bond energy, which is much higher than that of a vinylic C-H bond and very much higher than that of a secondary alkyl C-H bond: compare the dissociation energies (DH 298 in kcal/mol) for acetylene (131.3 ± 0.7), ethylene (109.8 ± 0.8), and the secondary C-H of propane (98.6 ± 0.4) (Blanksby and Ellison 2003;Ervin et al 1990).…”

Section: Oxidation Of Monosubstituted Acetylenes To Acetic Acid Derivmentioning

confidence: 99%

Acetylenes: cytochrome P450 oxidation and mechanism-based enzyme inactivation

Montellano

2019

Drug Metabolism Reviews

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The oxidation of carbon-carbon triple bonds by cytochrome P450 produces ketene metabolites that are hydrolyzed to acetic acid derivatives or are trapped by nucleophiles. In the special case of 17αethynyl sterols, D-ring expansion and de-ethynylation have been observed as competing pathways. The oxidation of acetylenic groups is also associated with mechanism-based inactivation of cytochrome P450 enzymes. One mechanism for this inactivation is reaction of the ketene metabolite with cytochrome P450 residues essential for substrate binding or catalysis. However, in the case of monosubstituted acetylenes, inactivation can also occur by addition of the oxidized acetylenic function to a nitrogen of the heme prosthetic group. This addition reaction is not mediated by the ketene metabolite, but rather occurs during oxygen transfer to the triple bond. In some instances, a detectable intermediate is formed that is most consistent with a ketocarbene-iron heme complex. This complex can progress to the N-alkylated heme or revert back to the unmodified enzyme. The ketocarbene complex may intervene in the formation of all the N-alkyl heme adducts, but is normally too unstable to be detected.

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“…Replacement of the * Author to whom correspondence should be addressed. acetylenic hydrogen of PA1 (4,5) and biphenylacetylene (4,5,11) with deuterium results in a kinetic isotope effect on product formation (Ah/Ad) between 1.4 and 1.8. This is a large isotope effect for a process in which the hydrogen migrates through a bent rather than linear transition state (14).…”

Section: With [2h]-and [13c]-labeledmentioning

confidence: 99%

“…The cytochrome P450-catalyzed oxidation of terminal acetylenes converts them to acetic acid derivatives and results in concomitant inactivation of the enzyme (1-3). Acetic acid derivatives have been explicitly identified in the metabolism of phenylacetylenes (4, 5), p-biphenylacetylenes (6)(7)(8)(9)(10)(11), 2-ethynylnaphthalene (12), and 10undecynoicacid (13). Studies…”

Section: Introductionmentioning

confidence: 99%

Determinants of protein modification versus heme alkylation: inactivation of cytochrome P450 1A1 by 1-ethynylpyrene and phenylacetylene

Wk¹,

Z²,

Pr³

1993

Chem. Res. Toxicol.

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Reaction of cytochrome P450 enzymes with arylacetylenes results in heme N-alkylation [e.g., Komives, E. A., and Ortiz de Montellano, P. R., (1985) J. Biol. Chem. 260, 3330-3336] and/or protein modification [e.g., Gan, L.-S. L., Acebo, A. L. and Alworth, W. L. (1984) Biochemistry 23, 3827-3836]. To clarify the factors that determine whether heme or protein alkylation occurs, we have investigated the cytochrome P450 1A1-catalyzed oxidation of 1-ethynylpyrene (1-EP) and phenylacetylene (PA). Cytochrome P450 1A1 in microsomes from beta-naphthoflavone-induced rats is inactivated in a time- and NADPH-dependent manner by 1-EP and PA. Parallel loss of the heme chromophore is observed with PA but not with 1-EP, although partial heme chromophore loss is observed when the purified, reconstituted enzyme is inactivated by either agent. Product analysis shows that 1-EP and PA are oxidized to, respectively, (1'-pyrenyl)-acetic and phenylacetic acids. In contrast to the inactivation of cytochrome P450 2B1 by PA, no isotope effect is observed on enzyme inactivation or metabolite formation when the acetylenic hydrogen is replaced by deuterium in either 1-EP or PA. Inactivation of cytochrome P450 1A1 by 1-EP results in covalent binding of 0.8-0.9 equiv (relative to total cytochrome P450 content) of the inhibitor to the microsomal protein. The results demonstrate that a single isozyme can be inactivated, depending on the structure of the arylacetylene, by heme or protein alkylation. Spectroscopic binding constants (Ks) show that 1-EP binds to the enzyme with > 2000 times greater affinity that PA.(ABSTRACT TRUNCATED AT 250 WORDS)

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Deuterium-isotope effect in the biotransformation of 4-ethynylbiphenyls to 4-biphenylylacetic acids by rat hepatic microsomes

Cited by 13 publications

References 5 publications

Characterization of Novel Glutathione Adducts of a Non-Nucleoside Reverse Transcriptase Inhibitor, (S)-6-Chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3,4-dihydro-2(1H)-quinazolinone (DPC 961), in Rats. Possible Formation of an Oxirene Metabolic Intermediate from a Disubstituted Alkyne

Characterization of Novel Glutathione Adducts of a Non-Nucleoside Reverse Transcriptase Inhibitor, (S)-6-Chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3,4-dihydro-2(1H)-quinazolinone (DPC 961), in Rats. Possible Formation of an Oxirene Metabolic Intermediate from a Disubstituted Alkyne

Acetylenes: cytochrome P450 oxidation and mechanism-based enzyme inactivation

Determinants of protein modification versus heme alkylation: inactivation of cytochrome P450 1A1 by 1-ethynylpyrene and phenylacetylene

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