Brian C. Challis

Oxidation of N-nitrosopiperidine in the Udenfriend model system and its metabolism by rat-liver microsomes.

Abstract The major product isolated from the oxidation of the carcinogen, N-nitrosopiperidine, by the ascorbic acid model system of Udenfriend et al. has been identified as N-nitroso-4-piperidone both by its spectral properties and by comparison with authentic material. On incubation of N-nitrosopiperidine with rat-liver microsomes in the presence of the appropriate co-factors, N-nitroso-4-hydroxypiperidine was the major metabolite. This was identified by comparison of its mass spectrum and chromatographic properties with those of N-nitroso-4-hydroxypiperidine obtained by nitrosation of 4-hydroxypiperidine. The significance of these products with respect to the mechanism of action of the parent carcinogen, N-nitrosopiperidine, is discussed.

The chemistry of nitroso-compounds. Part 11. Nitrosation of amines by the two-phase interaction of amines in solution with gaseous oxides of nitrogen

The formation of secondary N-nitrosoamines when MeCN solutions of amines are brought into contact with gaseous NO, N2O3, and N2O4 at 25 °C is reported. With NO, N-nitrosoamine formation from piperidine, morpholine, and diphenylamine occurs very slowly (t½ca. 8 days). Reaction rates are largely independent of the amine, suggesting that oxidation of NO by adventitious oxygen is the slow step. Very much faster reactions are observed, however, with N2O3 and N2O4. With a ca. 10-fold excess of N2O3 or N2O4, quantitative yields of both N-nitrosopiperidine and N-nitrosodiphenylamine are found in less than 3 min. With a 27-fold excess of piperidine and N2O4 only, ca. 8%N-nitropiperidine and 92%N-nitrosopiperidine are obtained. Rapid reactions also ensue when solutions of either primary aromatic or secondary amines dissolved in 0.1M-aqueous NaOH are brought into contact with gaseous N2O3 and N2O4. With a ca. 2–400-fold excess of these nitrogen oxides, 12–65% of the amine is converted either to N-nitrosoamine or diazonium ion in less than 3 min. Competitive hydrolysis of the nitrogen oxide by the solvent is not HO–-catalysed and the amine : H2O reactivities are in ca. 1 000 : 1. The extent of N-nitrosation varies insignificantly over a wide range of basicities (pKa, 11.12 to –1.0), but no reaction occurs with either 2,4-dinitroaniline or N-butyl-acetamide. With N2O4, smaller amounts of N-nitroamines form concurrently and increase with decreasing amine basicity. The results are discussed in relation to amine nitrosation by N2O3 and N2O4 in aqueous acidic solutions. It is suggested that the lower selectivity for the dissolved gaseous reagents may relate to the presence of more reactive N2O3 and N2O4 isomers. The results also show that carcinogenic N-nitrosoamines form under a much wider range of experimental conditions than previously known, some of which are relevant to atmospheric pollution.

The chemistry of nitroso compounds. Part 12. The mechanism of nitrosation and nitration of aqueous piperidine by gaseous dinitrogen tetraoxide and dinitrogen trioxide in aqueous alkaline solutions. Evidence for the existence of molecular isomers of dinitrogen tetraoxide and dinitrogen trioxide

Detailed quantitative results are reported for the interaction of aqueous piperidine in aqueous 0.1M-NaOH at 25° with gaseous N2O4 and N2O3. Both reagents rapidly give substantial amounts of N-nitrosopiperidine, plus smaller amounts of N-nitropiperidine in the case of N2O4, in addition to hydrolysis products such as NO2–. All these reactions are considered to occur predominantly in the aqueous phase and to be complete in a few seconds. With excess amine, yields of N-nitrosopiperidine reach maximum values corresponding to 100% for N2O3 but only ca. 50% for N2O4. The yield of N-nitropiperidine from N2O4, however, shows no maximum even at the highest [Piperidine]. The dependence of product yields on initial [Piperidine] and [N2Ox] suggests that N-nitrosopiperidine formation follows Rate =kp[Piperidine][N2Ox]. The concurrent hydrolysis of N2O3 and N2O4 is not significantly catalysed by HO– and is considered to involve H2O only. On a molar basis, piperidine is more reactive than H2O towards nitrosation by N2O3 and N2O4 by factors of 3 300 and 2 000, respectively. The results are discussed in relation to the existence of two molecular isomers for both N2O3 and N2O4, and the mechanisms by which these entities react with amines. For N2O4, the more stable symmetrical O2N–NO2 is considered to form only N-nitropiperidine, probably via a four-centre transition state: N-nitrosopiperidine results from concurrent reaction by the less stable ON–ONO2 isomer formed in aqueous solution by dimerisation of NO2 from the gaseous phase. For N2O3(which is fully dissociated in the gaseous phase) recombination of NO with NO2 in aqueous solution produces the less stable, symmetrical ON–ONO rather than the more stable ON–NO2 isomer present in aqueous HNO2. The existence of two isomers explains the higher reactivity of gaseous N2O3 towards weakly basic amines. N-Nitrosopiperidine formation with gaseous N2O3 results predominantly from nucleophilic attack by the amine on the ON–ONO isomer. Analysis of the data suggests that the formation of both ON–ONO2 and ONONO from their radical components in solution may be the rate-limiting step for the reactions leading to N-nitrosopiperidine.

The mutagenic properties of N-nitrosopeptides in the Ames test

N-(N-Acetyl-L-prolyl)-N-nitrosoglycine (APNG) and N-(N-acetylvalyl)-N-nitrosoglycine (AVNG) are shown to exert mutagenic activity in the Salmonella/mammalian microsome mutagenicity (Ames) test. Positive responses are apparent for base-pair substitution mutation-detecting strains (TA1535, TA100 and TA102) both with and without the addition of S9-mix. It is concluded that both APNG and AVNG are direct-acting mutagens.

The reaction of geminal bromonitroalkanes with nucleophiles. Part 1. The decomposition of 2-bromo-2-nitropropane-1,3-diol (‘Bronopol’) in aqueous base

2-Bromo-2-nitropropane-1,3-diol decomposes in aqueous base to give tris(hydroxymethyl)-nitromethane, glycolic acid, formic acid, methanol and 2,2-dinitroethanol. It also releases NO2– and Br– ions but not BrO–. These products are shown to form via four concurrent decomposition pathways, three of which involve 2-bromo-2-nitroethanol as a reactive intermediate.

Mutagenic properties of N-nitrosopeptides in mammalian cell culture assays

Two N-nitrosopeptides, N-(N-acetyl-L-prolyl)-N-nitrosoglycine and N-(N-acetylvalyl)-N-nitrosoglycine, were investigated for genetic toxicity towards mammalian cells using an established line of Chinese hamster ovary cells (CHO-K1-BH4). Observations were made on three indices of genetic damage, namely chromosome aberrations, sister chromatid exchange and induction of thioguanine-resistant variants. Treatment of cells with either compound resulted in dose-dependent increases in all indices, indicating that both compounds are direct-acting mutagens.

Synthesis and stability of N-nitrosodipeptides

The Synthesis, characterization, and chemical properties of the N-nitroso derivatives of some N-(N-acetylprolyl)-peptides are described.

Rapid nitrosation of amines in aqueous alkaline solutions by β-substituted alkyl nitrites

Alkyl nitrites bearing β-electron withdrawing substituents, either synthesized independently (e.g. 2-ethoxyethyl nitrite) or formed in situ by reaction between nitrosyl gases and an alcohol or carbohydrate group, effect the rapid nitrosation of basic secondary amines in 0·1 M NaOH at 25 °C.

Nitrosation under alkaline conditions

Both gaseous N2O3 and N2O4, but not NO, are shown to effect the nitrosation of primary and secondary amines in neutral and alkaline aqueous solutions; the reaction rates are rapid, insensitive to amine basicity and are not inhibited by HO–, which is consistent with radical pathways involving NO2 and NO, and therefore carcinogenic N-nitrosamines may form under a much wider range of experimental conditions than hitherto suspected.

The chemistry of nitroso-compounds. Part VII. The first ‘fast’ proton transfer for an aromatic nitrosation

Rates of nitrosation are reported for phenol and 2-naphthol in aqueous carboxylic acid buffers over the pH range 1–ca. 5·5 at 25 °C. Substitution of phenol occurs predominantly at the para-position at rates which are pH independent, but general-base catalysed, below pH ca. 4·5, and comparative experiments with [4-2H]phenol show the existence of a substantial primary hydrogen isotope effect (kH/kD= 3·5). Above pH ca. 4·5, however, the kinetic dependences for phenol show significant changes. These differences are more easily seen with 2-naphthol, which undergoes nitrosation exclusively at the 1-position. Here reaction rates are pH independent, subject to general-base catalysis, and to substantial primary hydrogen isotope effects (kH/kD= 4·0) only below pH ca. 2. At higher pH, the rate is proportional to [H3O+], and both abse catalysis and isotope effects diminish substanitally. All these observations are consistent with a common A–SE2 reaction mechanism in which proton expulsion from a dienone intermediate is rate limiting for phenol at pH ca. 4·0, so this is the first-known aromatic nitrosation for which proton expulsion is rapid. Neither nitrous anhydride (N2O3) nor nitrosyl acetate (NOOAc) is sufficiently reactive to substitute the nucleus of phenol or 2-naphthol and reaction at pH 1–ca. 5 probably involves the nitrous acidium ion (H2NO2+); with halide ions present, additional reaction via nitrosyl halides occurs. The pH dependent rate for 2-naphthol excludes significant nitrosation of the 2-naphtholate ion up to pH ca. 5, but this species may preferentially interact at oxygen to form an unstable aryl nitrite. The implications of this deduction on the ambident nucleophilic properties of phenolic compounds, and the incursion of free radical substitution pathways, are also discussed.