What Makes a Potent Nitrosamine? Statistical Validation of Expert-Derived Structure–Activity Relationships

Abstract

The discovery of carcinogenic nitrosamine impurities above the safe limits in pharmaceuticals has led to an urgent need to develop methods for extending structure−activity relationship (SAR) analyses from relatively limited datasets, while the level of confidence required in that SAR indicates that there is significant value in investigating the effect of individual substructural features in a statistically robust manner. This is a challenging exercise to perform on a small dataset, since in practice, compounds contain a mixture of different features, which may confound both expert SAR and statistical quantitative structure− activity relationship (QSAR) methods. Isolating the effects of a single structural feature is made difficult due to the confounding effects of other functionality as well as issues relating to determining statistical significance in cases of concurrent statistical tests of a large number of potential variables with a small dataset; a nai ̈ve QSAR model does not predict any features to be significant after correction for multiple testing. We propose a variation on Bayesian multiple linear regression to estimate the effects of each feature simultaneously yet independently, taking into account the combinations of features present in the dataset and reducing the impact of multiple testing, showing that some features have a statistically significant impact. This method can be used to provide statistically robust validation of expert SAR approaches to the differences in potency between different structural groupings of nitrosamines. Structural features that lead to the highest and lowest carcinogenic potency can be isolated using this method, and novel nitrosamine compounds can be assigned into potency categories with high accuracy.

Strategies for Assessing Acceptable Intakes for Novel N-Nitrosamines Derived from Active Pharmaceutical Ingredients

“…Small alkyl groups (e.g., methyl and ethyl) next to the N -nitrosamine center are associated with high carcinogenic potency in animals ,, as well as being generally associated with positive prevalence as genotoxins. Furthermore, the archetypical N -nitrosamines such as NDEA and NDMA, as well as other structures presented in Table such as NMBA, contain methyl or ethyl substituents.…”

“…It is worth noting that in the case of some if not all of these structural attributes, the presence of two features that act in the same direction will lead to synergistic effects. For example, it is observed that all N -nitrosamines with two strong β-electron-withdrawing groups are negative whereas those with only one are weakly carcinogenic or that the set of three compounds NDMA, NDEA, and N -ethyl- N -methylnitrosamine (with small unsubstituted alkyl substituents flanking the N -nitroso group) are positive and potent animal carcinogens, whereas having a single ethyl or methyl group and another, larger, substituent, while leading generally to positive results, does not in general show the same association with high potency. As a counterpoint, when an N -nitrosamine contains structural features from both lists such as NMBA (carboxylic acid and methyl side chain), the effects of the features can, pragmatically, be thought to “cancel” and the N -nitrosamine is likely to be of a level of concern comparable to the class mean rather than to the potent rodent carcinogen NDMA.…”

“…Strong β-EWGs reduce the electron density on the carbon α to both the nitrosamine and the EWG via induction. This attribute decreases the propensity toward CYP-mediated α-carbon oxidation, , since a potential partial charge on the already δ + carbon is strongly disfavored; this feature moves the required transition state to be of higher energy, slowing the reaction . Experimentally, relative to NDEA, N -nitroso- N -(2,2,2-trifluoroethyl)­ethylamine is a weak carcinogen, whereas N -nitroso- N , N -bis­(2,2,2-trifluoroethyl)­amine is non-carcinogenic (Figure ).…”

“…In conclusion, available information regarding structural features that govern N -nitrosamine carcinogenicity in animals suggest an important trend; substituents which negatively impact the CYP-mediated rate-limiting α-carbon hydroxylation on the N -nitrosamine, either electronically or via steric hindrance, are expected to result in a reduction in the carcinogenic potency, which can be taken into account when projecting daily AIs for novel N -nitrosamines using the read-across methodology described in section .…”

A Quantum-Mechanical Approach to Predicting Carcinogenic Potency of N-nitrosamine Impurities in Pharmaceuticals

“…4 However, direct use of SARs in predicting potency of N-nitrosamines is impractical due to the many confounding factors, the sheer complexity of the biochemical process and the limited dataset of reliable TD50's to train such models. 4,78 On the other hand, the use of QSARs based on physicochemical properties can be grossly misleading given a dubious link between these descriptors and the mechanism of action. Since covalent interactions dominate Nnitrosamine metabolism, the solution appears to be in the development of robust quantummechanical approaches based on electronic structure.…”