Module 3 - Strategic case studies in practice

2.4.2.1. Reactive metabolites

N- Nitrosamines need to be activated metabolically to form different diazonium ions (e.g. methyldiazonium, ethyldiazonium etc.). Alkydiazonium ions are precursors of reactive electrophilic carbenium ions, which directly react with DNA thereby forming stable adducts mainly with nitrogen and oxygen of guanine, cytosine and thymidine. The structure and number of diazonium ions formed from a specific N- nitrosamine depends on the chemical structure of each individual N- nitrosamine. The resulting DNA-adducts depend on the nature of the formed diazonium ion. These different adducts are repaired by different cellular repair mechanisms with different capacity, velocity and accuracy. For the high mutagenic and carcinogenic potential of some volatile N- nitrosamines, the key step is metabolic activation by α -hydroxylation which subsequently leads to formation of diazonium ions. Lower number of α –hydrogens in alkyl- N- nitrosamines and substitution of the α -hydrogen may reduce the mutagenic potential. This consideration would lead to a ranking of the relative mutagenic potency of alkyl- N- nitrosamines: dimethyl, diethyl…> methyl nitroso…butanoic acid (one α –hydrogen)…>> tertiary-butyl (no α -hydrogen). Other substituents may also reduce or even eliminate the mutagenic and carcinogenic potential of N- nitrosamines such as branched, bulky or un-metabolisable groups at or near the α -carbon preventing metabolic activation [Benigni et al. (2005)]. Another factor for mutagenic potency is the stability of the diazonium ion formed. Usually, the stabilizing effect of the substituent is expected to decrease in the order isopropyl >carboxypropyl> ethyl > methyl, resulting in thermodynamic stabilities as follows: isopropyldiazonium ion >carboxypropyldiazonium ion >ethyldiazonium ion >methyldiazonium ion. Assuming a nucleophilic substitution 2 (Sn2) reaction mechanism for alkylation of DNA by alkyldiazonium ions, steric hindrance has to be taken into account as an additional factor in reactivity estimation. Accordingly, kinetic reactivity of alkyldiazonium ions towards nucleophils via Sn2 reaction is expected to increase as follows: isopropyl diazonium ion

Figure 2.4.2.1-1 Relative alkylation rates / hydrolysis rates for alkyldiazonium ions [Manso et al. (2008)].

EMA/369136/2020

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