3-Hydroperoxy-N-nitrosomorpholine in buffered aqueous media in the presence of calf thymus DNA was treated with a phosphine reductant to generate the transient α-hydroxynitrosamine and subsequent diazonium ion that alkylated the DNA, as previously reported. Subsequent addition of hydride donors, for 30 min, followed by acid hydrolysis of the mixture allowed detection and quantification of 6-(2-(2-((9H-purin-6-yl)amino)ethoxy)ethoxy)-9H-purin-2-amine, the reduced cross-link formed from deposition, via the diazonium ion, of a 3-oxa-pentanal fragment on O6-Gua, and condensation with N6-Ade, presumably in the vicinity. Decreasing temperature of the reactions and decreasing pH modestly increased the yields of trapped crosslink. Among three borohydride reductants, NaNCBH3 is superior, being ∼4 times more effective on a molar basis, as opposed to a hydride equivalent basis, than NaBH4 or Na(AcO)3BH. For trapping with NaNCBH3, it is deduced that the reaction likely occurs with the iminium ion that is in protonic equilibrium with its conjugate base imine. In an experiment in which the hydroperoxide was decomposed and NaNCBH3 was introduced after various times, the amount of cross-link was observed to increase, nearly linearly, by about four-fold over one week. These data indicate that there are a minimum of 2 populations of cross-links, one that forms rapidly, in minutes, and another that grows in with time, over days. Reduced nicotinamide co-factors and ascorbate are observed to effect reduction (over 3 days) of the cross-links confirming the possibility that otherwise reversible cross-links might be immortalized under biological conditions.

1,4-Dioxan-2-one, 1, was synthesized and the equilibrium constant between it and the hydrolysis product 2-(2-hydroxyethoxy) acetic acid, 2, was determined as KO = 70 ± 3.5 in acidic aqueous media, 25 °C, ionic strength 1M, (KCl), 5% by volume acetonitrile. The carboxylic acid dissociation constant of 2 was determined under the same conditions to be pKa = 3.31 ± 0.02. On the basis of these two determinations, the equilibrium constant between 1 and carboxylic acid anion, 3, and the proton was calculated to be KOA = 0.034 ± 0.002 M. The stability of 1 was determined in the range of pH between 1 and 8.5 in buffered aqueous solutions under the conditions above by UV spectrophotometric methods and exhibited simple first order kinetics of decay. On the basis of buffer dilution plots, the values of ko, the rate constant for solvent mediated decomposition, were determined. The plot of log ko against pH is consistent with a three term rate law for solvolysis with a hydrogen ion catalyzed rate constant kH+ = 1.1 (± 0.1) M−1 min−1, a water catalyzed rate constant, kw = 9.9 (± 0.5) × 10−4 min−1, and a hydroxide ion catalyzed rate constant, kOH = 4.1 (± 0.3) × 104 M−1 min−1. The t1/2 for decay at pH = 7.0, at 25 °C, is ~2 h. Treatment of F344 rats with aflatoxin B1 (AFB1) (positive control) elicited the expected preneoplastic foci in the livers of each rat tested, while subsequent administration of 1 (a total of 1.32 g over 12 weeks) failed to statistically increase focal number or focal volume percent. In 8 rats administered 1 (1.32 g, 12 weeks) alone, no increase above background foci was detected. This study does not support compound 1 as a common carcinogen.

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