Formation Of Nitrenium Ion Research Articles

Novel Identification of Cysteinyl Derivatives of Toxic Clozapine Nitrenium Ions and the Effect of Valproic Acid on Metabolite Formation: A Study Using Reprocessed High-Resolution Mass Spectra of Analyzed Therapeutic Drug Monitoring Samples.

Clozapine (CLZ) use is hampered by the risk of granulocyte toxicity, which is associated with the formation of nitrenium ions and the concurrent use of valproic acid (VPA). These highly reactive nitrenium ions cannot be measured in vivo. Instead, deactivated cysteinyl conjugates may potentially be detected. The aim of this study was to develop a novel method for identifying cysteinylated derivates of CLZ nitrenium ions to investigate the effect of VPA on their formation using therapeutic drug monitoring data. A population comprising 93 VPA comedicated and 162 control patients from a therapeutic drug monitoring (TDM) service in Oslo, Norway, was included. Reprocessing of ultraperformance liquid chromatography high-resolution mass spectra (UHPLC-HR-MS) of previously analyzed TDM samples, combined with the assessment of MS/MS fragmentation patterns, was performed to identify the CLZ cysteinyl conjugates. Smoking, which induces CLZ metabolism, was assessed by detecting cotinine in the reprocessed mass spectra. By reprocessing the UHPLC-HR-MS files of the TDM analyses and reviewing the MS/MS fragment profiles, four cysteinyl conjugates of CLZ were identified. The formations of CLZ cysteinyl (CLZ-Cys 1+ ) and CLZ- N -oxide cysteinyl (CLZ-NOX-Cys 1+ ) were 1.5-fold ( P = 0.038) and 2.1-fold ( P < 0.001) higher in VPA-treated patients than those in the controls. In agreement with previous studies, a 45% reduction in N -desmethylclozapine formation was observed in VPA-treated patients ( P < 0.001). A novel method for detecting cysteinyl conjugates of CLZ was developed. Application of this method indicated that VPA significantly increased the formation of CLZ-Cys 1+ metabolites, which might explain the granulocyte toxicity reported after adding VPA to CLZ treatment. The developed method opens new avenues for investigating CLZ toxicity, e.g. by correlating cysteinyl conjugates and granulocyte counts in patients.

Soft-Hard Acid/Base-Controlled, Oxidative, N-Selective Arylation of Sulfonanilides via a Nitrenium Ion.

In iodine(III)-catalyzed, dehydrogenative arylations of sulfonanilides, the functionalization of C-C bonds is preferred over the functionalization of C-N bonds. Herein, an unprecedented N-selective arylation of sulfonanilides using soft-hard acid-base (SHAB) control by a nitrenium ion over a carbenium ion is reported. Treatment of sulfonanilides with iodine(III) led to the formation of nitrenium ions (soft), which preferentially react with biphenyls (soft) over bimesityl (hard) to generate C-N bonds. The iodine(III) was generated in situ by using PhI and mCPBA at room temperature.

Theoretical Studies of the Mechanism of N-Hydroxylation of Primary Aromatic Amines by Cytochrome P450 1A2: Radicaloid or Anionic?

Primary aromatic and heteroaromatic amines are notoriously known as potential mutagens and carcinogens. The major event of the mechanism of their mutagenicity is N-hydroxylation by P450 enzymes, primarily P450 1A2 (CYP1A2), which leads to the formation of nitrenium ions that covalently modify nucleobases of DNA. Energy profiles of the NH bond activation steps of two possible mechanisms of N-hydroxylation of a number of aromatic amines by CYP1A2, radicaloid and anionic, are studied by dispersion-corrected DFT calculations. The classical radicaloid mechanism is mediated by H-atom transfer to the electrophilic ferryl-oxo intermediate of the P450 catalytic cycle (called Compound I or Cpd I), whereas the alternative anionic mechanism involves proton transfer to the preceding nucleophilic ferrous-peroxo species. The key structural features of the catalytic site of human CYP1A2 revealed by X-ray crystallography are maintained in calculations. The obtained DFT reaction profiles and additional calculations that account for nondynamical electron correlation suggest that Cpd I has higher thermodynamic drive to activate aromatic amines than the ferrous-peroxo species. Nevertheless, the anionic mechanism is demonstrated to be consistent with a variety of experimental observations. Thus, energy of the proton transfer from aromatic amines to the ferrous-peroxo dianion splits aromatic amines into two classes with different mutagenicity mechanisms. Favorable or slightly unfavorable barrier-free proton transfer is inherent in compounds that undergo nitrenium ion mediated mutagenicity. Monocyclic electron-rich aromatic amines that do not follow this mutagenicity mechanism show significantly unfavorable proton transfer. Feasibility of the entire anionic mechanism is demonstrated by favorable Gibbs energy profiles of both chemical steps, NH bond activation, and NO bond formation. Taken together, results suggest that the N-hydroxylation of aromatic amines in CYP1A2 undergoes the anionic mechanism. Possible reasons for the apparent inability of Cpd I to activate aromatic amines in CYP1A2 are discussed.

Potential Role of a Quetiapine Metabolite in Quetiapine-Induced Neutropenia and Agranulocytosis

Clozapine was the first of the atypical antipsychotics to be developed, but its use has been restricted because of toxicity issues, particularly the risk of potentially life-threatening drug-induced neutropenia and agranulocytosis, which occurs in about 1% of patients. Bioactivation of clozapine by peroxidases forms a reactive nitrenium ion, which covalently adducts to protein and leads to neutrophil toxicity. The current generation of clozapine-inspired atypical antipsychotics has reduced toxicity through improved potency/decreased dose or through structural modification to prevent peroxidase-catalyzed nitrenium ion formation. Through the substitution of sulfur for the bridging nitrogen found in clozapine, quetiapine does not directly form a nitrenium ion when incubated with myeloperoxidase/H(2)O(2). We present evidence that cytochrome P450 2D6 catalyzes the formation of 7-hydroxyquetiapine, which can be oxidized by human myeloperoxidase to form a reactive quinone-imine and a reactive radical, which may account for the continued, although reduced, neutrophil toxicity. In the presence of myeloperoxidase/H(2)O(2) and glutathione, covalent 7-hydroxyquetiapine-glutathione adducts were formed. Bioactivation of quetiapine was verified in vivo in rat where three 7-hydroxyquetiapine-mercaptate adducts and a 7-hydroxyquetiapine-glutathione adduct were detected from bile after oral dosing. The decreased incidence of agranulocytosis with quetiapine over clozapine is postulated to be due to the lower exposure of the toxic precursor, 7-hydroxyquetiapine versus clozapine, respectively.

Interaction of clozapine and its nitrenium ion with rat D2 dopamine receptors: in vitro binding and computational study

The interaction of diazepine analogues like clozapine or olanzapine with D2 receptor was greatly affected by a mixture of HRP/H(2)O(2) known to induce the formation of nitrenium ion. Unlike diazepine derivatives, the oxidative mixture had low impact on the affinity of oxa- and thiazepine derivatives such as loxapine, clothiapine or JL13 for the D2 receptor. Molecular docking simulations revealed a huge difference between the mode of interaction of clozapine nitrenium ion and the parent drug. Electronic and geometric changes of the tricyclic ring system caused by the oxidation appeared to prevent the compound finding the correct binding mode and could therefore explain the difference observed in binding affinities.

Ultimate Carcinogenic Metabolites from Aromatic and Heterocyclic Aromatic Amines: A Computational Study in Relation to Their Mutagenic Potency

The formation of nitrenium ions from their precursors was examined by density functional theory (DFT) calculations in order to analyze the role of these electrophilic intermediates on the mutagenic activity of the parent amines. The relative reactivities for N-O bond dissociation from the N-hydroxy, N-acetoxy and N-sulfate derivatives of aniline were evaluated. Furthermore, the N-acetoxy esters from a set of 17 aromatic and heteroaromatic amines of diverse structure were considered, and correlations were sought between the calculated properties and the reported mutagenic potencies. The mutagenic activity was found to increase when a more negative charge developed at the exocyclic nitrogen of the nitrenium ion (qN) and with nitrenium ion stability. Different functional correlations were observed for the amine derivatives grouped according to their classification as aromatic (Ar), imidazo-carbocyclic (Imi-C), and imidazo-heterocyclic (Imi-H). The formation of N-acetyl nitrenium ions from aromatic amides was also considered and found to be less favorable than nitrenium ion generation from the corresponding amines.

NMR Spectroscopy and Molecular Orbital Calculations to Interpret the Mutagenicity of Nitrobenzanthrones

Thorough NMR measurements were made on 3-nitro-7H-benz[d,e]anthracen-7-one (1), 1,9-dinitro-7H-benz[d,e]anthracen-7-one (2), 3,11-dinitro-7H-benz[d,e]anthracen-7-one (3), and 11-nitro-7H-benz[d,e]anthracen-7-one (4), whose mutagenic activities vary largely with the position and the number of the nitro group. Their 1H and 13C chemical shifts were assigned by means of several kinds of two dimensional NMR spectroscopic techniques, and correlations were examined between the chemical shifts and mutagenicity among the compounds. The chemical shifts at the ortho carbon atoms with respect to the nitro group differ between mutagenic compounds (1) – (3) and nonmutagenic compound (4). The difference can be related to that in the reactivity toward formation of nitrenium ions by taking into account of the electron density on the ortho carbon atoms obtained by molecular orbital calculations.

Metabolic activation of carcinogenic 1-nitropyrene by human cytochrome P450 1B1 in Salmonella typhimurium strain expressing an O-acetyltransferase in SOS/ umu assay

Metabolic activation of 1-nitropyrene (1-NP) by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes was investigated. 1-NP induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of any P450 system, but the activities were influenced by the levels of bacterial O-acetyltransferase (OAT) and nitroreductase. Metabolic activation of 1-NP by human P450 1B1/NPR membranes was observed and was influenced by the levels of OAT levels in tester strains. Metabolic activation of 1-NP (0.3 μM) by P450 1B1 was 750 umu units/min/nmol P450 1B1 in an OAT-overexpressing strain NM2009. The metabolic activation of 1-NP (3–30 μM) was similar (∼300 umu units/min/nmol P450 1B1) using TA1535/pSK1002 or OAT-deficient strain NM2000. P450 1B1 had the highest catalytic activities among P450 family 1 enzymes for the activation of 1-aminopyrene (1-AP) in the OAT-overexpressing strain NM2009, suggesting nitrenium ion formation via N-hydroxylation/ O-acetylation. High-performance liquid chromatography (HPLC) analyses revealed the formation of 1-nitropyrene-6-ol and also 1-nitropyrene-3-ol, 1-nitropyrene-8-ol, and trans-4,5-dihydroxy-4,5-diol-1-nitropyrene from 1-NP (10 μM), catalyzed by P450 1B1. These results indicate that 1-NP can be activated by human P450 1B1 to a genotoxic agent by nitroreduction/ O-acetylation at low substrate concentrations and probably by epoxidation (independent of OAT) at high concentrations.

Relative stabilities of nitrenium ions derived from polycyclic aromatic amines. Relationship to mutagenicity

The relative energetics of arylamine N-hydroxylation and N-O heterolysis (ArNH 2 → ArNHOH → ArNH +) for condensed systems of two, three and four rings were calculated using semiempirical AM1 molecular orbital theory. The overall thermodynamics of N-hydroxylation were almost insensitive to the structure of the amine while differences in the energetics of nitrenium ion formation varied from 0 to 35 kcal mol −1. Limited correlations between the latter and the experimental TA98 and TA100 mutagenicities of the amines are presented.

The solvolysis of N-acetoxy-4-acetylaminostilbene: Irreversible formation of nitrenium ions

Extensive kinetic studies and product analysis, coupled with 0–18 labeling experiments have shown that the solvolysis of N-acetoxy-4-acetylaminostilbene in buffered aqueous acetone proceeds via irreversible nitrenium ion formation. The conflict between this conclusion and that of a previous study is explained

The solvolysis of N-acetoxy-1-N-acetylaminonaphthalene and N-acetoxy-2-N-acetylaminonaphthalene: divergent chemistry and mutagenic activity

Upon solvolysis in neutral 40% aqueous acetone, N-acetoxy-1-N- and N-acetoxy-2-N-acetylaminonaphthalene exhibit completely different modes of reaction; the former reacts with nitrenium ion formation, while the latter undergoes acyl oxygen scission.

The solvolysis of N-acetoxy-2-acetylaminofluorene and N-acetoxy-4-acetylaminobiphenyl: delicate balance between nitrenium ion formation and hydrolysis

The solvolysis of N-acetoxy-2-acetylaminofluorene in aqueous acetone at neutral pH proceeds exclusively with nitrenium ion formation while under the same conditions, the 4-aminobiphenyl analogue exclusive acyl–oxygen scission.

Prostaglandin H synthase-mediated reaction of carcinogenic arylamines with tRNA and homopolyribonucleotides

Prostaglandin H synthase mediates the reaction of an extensive series of carcinogenic arylamines with tRNA. Structure-activity relationships suggest that benzidine is especially reactive due to extended conjugation between the 4,4'-diamino groups. In trapping experiments with homopolyribonucleotides, benzidine reacts with polyguanylic acid but 4-aminobiphenyl reacts with polycytidylic acid. The nitrenium ion of 4-aminobiphenyl (formed by N,O-acyltransferase activation of N-hydroxy-4-acetylaminobiphenyl) reacts primarily with polyguanylic acid and to a lesser extent with polyadenylic acid. The results suggest that arylamine activation by prostaglandin H synthase does not involve nitrenium ion formation.