Structure–activity considerations and in vitro approaches to assess the genotoxicity of 19 methane-, benzene- and toluenesulfonic acid esters

Abstract

Sulfonic acid esters are considered as potentially alkylating agents that may exert genotoxic effects in bacterial and mammalian cell systems. One possible source of human exposure stems from drug synthesis when the salt-forming agents methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid are used together with alcoholic solvents such as methanol, ethanol and propanol. In this study computer-assisted structural considerations and in vitro approaches (Ames mutagenicity test using Salmonella typhimurium strains TA98 and TA100, and the micronucleus test using L5178Y mouse lymphoma cells) were used to assess the genotoxic properties of 19 sulfonic esters. While all esters may be principally active as genotoxicants based on the presence of the sulfonate moiety, the statistical correlative multiple computer automated structure evaluation (MCASE) system (MC4PC version 1.0) using the Ames mutagenicity A2I module (version 1.54), rank-ordered the activity of the benzenesulfonic acid esters in the Ames test negligible due to an inactivating modulator and a deactivating fragment, whereas the methane- and toluenesulfonic acid esters were predicted to be positive in this test.

In the Ames test, with the exception of the p-toluenesulfonic acid ethyl and iso-butyl esters, all compounds came out positive in Salmonella strain TA100. Methanesulfonic iso-propyl, sec-butyl and benzenesulfonic acid iso-propyl ester also showed mutagenic potential in strain TA98. In general, differences between results seen in Ames tests performed with or without metabolic activation were rather small.

In L5178Y mouse lymphoma cells, benzenesulfonic acid n- and iso-butyl ester and p-toluenesulfonic acid iso-butyl ester did not increase the number of cells containing micronuclei. The other esters were positive in this micronucleus test; however, methanesulfonic acid iso-butyl ester was found to be only weakly positive at excessively cytotoxic concentrations. These compounds were generally found to be more potent with regard to micronucleus induction when tested without metabolic activation (20 h treatment).

In conclusion, the iso-propyl esters of the three sulfonic acids under study were found to be the strongest mutagens, either when tested in the Ames test or the micronucleus assay, whereas p-toluenesulfonic acid iso-butyl ester was the only compound shown to be devoid of a genotoxic potential in both tests.

Introduction

The synthesis of new pharmaceutical entities involves the use of reactive substances that, as expected from their chemical properties, may show genotoxic activity in short-term in vitro genotoxicity assays. Such compounds, if not efficiently purged during the process, may find their way into the final synthesis step, i.e. towards the (normally non-genotoxic) drug substance. Hence, their presence may need to be controlled at a very low level. An internal evaluation of Novartis’ synthesis schemes for active pharmaceutical ingredients in 2002 and 2003 for the presence of genotoxic intermediates demonstrated that genotoxic compounds appear in a significant percentage of all drug substance syntheses at some stage during the synthesis. Genotoxicity testing of these drug substances, however, yielded mostly negative results. Despite such negative tests, there may be a need for control of genotoxic intermediates that cannot be efficiently removed from the final product since they may constitute an undesirable carcinogenic hazard in case of human use.

Since organic chemistry involves the use of reactive chemicals, it is obvious that the presence of genotoxic substances in synthesis pathways is not totally avoidable. Sulfonic acid esters, for example, which are considered to be potentially alkylating agents, are likely formed during drug synthesis when the salt-forming agents, methanesulfonic acid (mesylate), benzenesulfonic acid (besylate) or p-toluenesulfonic acid (tosylate) are used together with alcoholic solvents like methanol, ethanol or propanol. Methyl methanesulfonate (CAS 66-27-3) and ethyl methanesulfonate (CAS 62-50-0) are well-known mutagenic impurities, of which the lowest observed effect concentrations (LOEC) in Salmonella strain TA100 are 5 and 1500 μg/plate, respectively (data not shown). Moreover, methyl methanesulfonate and ethyl methanesulfonate have both been demonstrated to be carcinogenic in animals and are suspected human carcinogens [1]. Despite the wide use of mesylate, besylate and tosylate salts as pharmaceuticals, literature data on genotoxic or carcinogenic properties of their esters are sparse.

In this study, computer-aided structure–activity reasoning and in vitro approaches are used to assess the genotoxic properties of 19 sulfonic esters with the aim to discriminate between active versus inactive, non-genotoxic sulfonic acid esters, which could consequently influence the use of salt-forming agents and alcohols during the drug synthesis.