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Organic Process Research & Development
Boronic Acids and Derivatives - Probing the Structure-Activity Relationships for Mutagenicity
Marvin M. Hansen,*,§ Robert A. Jolly,*,‡ Ryan J. Linder§
§
Small Molecule Design and Development, Lilly Research Laboratories, Eli Lilly and Company,
Lilly Corporate Center, Indianapolis, Indiana 46285, USA ‡
Health/Safety/Environmental, Lilly Research Laboratories, Eli Lilly and Company, Lilly
Corporate Center, Indianapolis, Indiana 46285, USA
Corresponding Authors *Email:
[email protected],
[email protected] ACS Paragon Plus Environment
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TOC GRAPHIC
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ABSTRACT Despite a recent publication describing boronic acids as a “novel class of bacterial mutagen,” process and analytical chemists may not be aware of the potential worker exposure hazards, or of the need to assess boron compounds as potential genotoxic impurities (GTIs) per ICH M7. This publication provides new Ames data for 44 commercially available boronic acids, boronic acid derivatives, and boron containing reagents. Trends in the Ames data are discussed from a structure-activity perspective. Common reagents such as bis(pinacolato)diboron and bisboronic acid were shown to be mutagenic in the Ames assay. Currently available in silico computational models were found to provide little value in predicting the outcome of the Ames assay for boronic acids and derivatives. We propose oxygen mediated oxidation of boron compounds to generate organic radicals as a potential mechanism for mutagenicity. It is hoped that this paper will result in increased awareness of this class of GTIs, and prompt publication of additional Ames data for boronic acids and their derivatives that will lead to improved (Q)SAR models, and an understanding of the mechanism of mutagenicity.
KEY WORDS GTI, mutagen, Ames, boron, boronic acid, (Q)SAR
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INTRODUCTION Boronic acids and their derivatives are one of the most common intermediates in organic synthesis.1 Driven by the seminal work of Suzuki and Miyaura,2 these building blocks have become “the ‘gold standard’ for biaryl construction, arguably resulting in the ubiquity of this moiety in medicinal chemistry.”3 Over the past decade large-scale application of the SuzukiMiyaura reaction for making pharmaceuticals has also become routine.4 Other common uses of boronic acids in organic synthesis include the emerging area of “boronic acid catalysis”,5 in particular “green” amide bond formation,5c and asymmetric addition to electron deficient alkenes.6 Part of the desirability of boronic acid reagents is their perceived general low toxicity,5 but a 2011 toxicology paper describes boronic acids as a “novel class of bacterial mutagen.”7 A series of boronic acids and esters were tested in the Ames assay8,9 in this report, and 12 of 13 compounds tested were found to be mutagenic. Since the mechanism of mutagenicity was not able to be determined in this initial study, a recommendation was made to control boronic acids in the synthesis of pharmaceutical agents to the levels accepted for other GTIs. Implementation of control strategies for GTIs in pharmaceuticals has been evolving for several years,10 and recently resulted in publication of the ICH M7 regulatory guidance.11 With growing awareness within the pharmaceutical industry of the potential for boronic acids and derivatives to be genotoxic, a survey of several pharmaceutical companies provided additional data showing that 14 of 17 proprietary boronic acid derivatives tested in the Ames assay were positive for mutagenicity.12 The percent Ames-positive results within the boronic acid derivatives structural class (82.4%) exceeded two structural classes more commonly recognized by process chemists as Ashby alerts13 and potentially genotoxic: alkylating agents (57.7%) and aromatic amines
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Organic Process Research & Development
(37.0%).
While the potential of boronic acids and derivatives to persist as GTIs in
pharmaceutical agents is of broad importance for process chemists, the potential genotoxicity of boron-containing therapeutic agents is of more direct medicinal relevance. An assessment of five boron containing therapeutic agents found no genetic toxicology liability.14 Bortezomib, a dipeptide boronic acid therapeutic agent, has also been reported to be Ames-negative.15 In addition, a study of nine boronic acids which are positive in the Ames assay did not find a genotoxic liability in eukaryotes.16 The toxicological assessment of boronic acid therapeutic agents falls to medicinal chemistry and toxicology teams. However, process and analytical chemists have a responsibility to work with their toxicology and risk assessment colleagues to evaluate the safety of boronic acids from a worker exposure perspective,17 and as potential GTIs in active pharmaceutical ingredients (APIs). Despite the growing attention within the toxicology community toward this new alert class for mutagenicity, awareness within the process and analytical chemistry community is lacking. In addition, the data reported to date suggests that the majority of boronic acids and derivatives are likely to be mutagenic, without enough reported data to build a dependable SAR (structure activity relationship) understanding or suitable computational in silico models for (Q)SAR (quantitative structure activity relationship) mutagenicity prediction.18 In order to address the lack of reported Ames data for this new alert class, we now report Ames data for 44 boronic acids and their derivatives, along with a comparison of the Ames data with available (Q)SAR predictions using commercially available in silico models.
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MATERIALS AND METHODS Selection of Boronic Acids and Derivatives Selection of compounds for Ames testing and (Q)SAR analysis was guided by the following principles: 1) commercially available, non-proprietary compounds, 2) comparison of boronic acids with the corresponding pinacol boronic ester19 when possible, 3) comparison of six derivatives of phenyl boronic acid, the simplest aryl boronic acid, 4) focus on aryl boronic acids rather than the less common alkyl boronic acids, 5) coverage of electron rich and electron poor aryl boronic acids, along with heterocyclic aryl boronic acids, and aryl groups with different steric environments around the boron. The test compounds were ordered from commercial catalog vendors and used as received.20
Ames Assay Methods Ames assays were conducted under contract at BioReliance (Rockville, MD) or at Covance (Greenfield, IN).
Standard plate incorporation assays were performed using either 2-strain
[Salmonella typhimurium (Sal) strains TA98 and TA100] or 5-strain [Sal strains TA98, TA100, TA1535, TA1537 and Escherichia coli (E. coli) strain WP2uvrA/pKM101] according to published methods.9, 21 Tests were performed at concentrations from 1.5 to 5000 mcg per plate in the absence and presence of liver S9 from Aroclor 1254 treated rats. All test compounds were dissolved in DMSO and diluted with agarose gel. After applying to the bacterial cell plates, the concentration was
