Synthesis, Mutagenicity, and Metabolism of Substituted 4,4'-Aminoakoxyazobenzene dyes

Abstract

This study is an extension of previous work in our laboratories pertaining to the effects of substituents on the mutagenicity of aminoazobenzene-based dyes. The previous work included a study aimed at exploiting the ability of bulky alkoxy groups to reduce the mutagenic potential of aminoazobenzene dyes, which unexpectedly revealed that 4-((3-(2-hydroxyethoxy-4-amino)phenylazo) N,N-bis(2-hydroxyethyl)aniline (dye 80) was among the more mutagenic dyes in that study, despite having non-mutagenic amines as reductive-cleavage products. The present study was undertaken to unveil the basis for the mutagenic activity of dye 80. To accomplish this goal, a group of substituted 4,4’ –diaminoazobenzene dyes (80, 89-92) was synthesized and their structures were characterized using 1H NMR, TOF-LC-ESI mass spectrometry, and combustion analysis. The purity of each dye was shown by HPLC to be 98-100 %. These new compounds were designed as tests of our hypothesis which stated that the mutagenicity of dye 80 arises from the metabolic cleavage of N-hydroxyethyl groups to give 4-((3-(2-hydroxyethoxy)-4-amino)phenylazo) N-(2-hydroxyethyl)-aniline (dyes 89) and 4-((3-(2-hydroxyethoxy-4-amino)phenylazo)aniline (dye 90) as direct-acting mutagens. 4-((3-(2-hydroxyethoxy-4-amino)phenylazo) N,N-bis-(3-hydroxypropyl)aniline (dye 91) arises from lengthening the N-alkyl groups of dye 80 from 2 to 3 carbons, while 4-((3-(2-hydroxyethoxy-4-amino)phenylazo)-N,N-bis(2-acetoxyethyl)aniline (dye 92) is a capped form of dye 80.

Mutagenicity was determined using the standard Ames test in Salmonella strains TA98, TA100, and TA1538 with and without S9 enzyme activation. The results showed that all of the dyes tested were mutagenic at various levels with and without S9 enzyme activation in TA1538. Dye 90 was also mutagenic in TA98 and TA100 with and without S9 enzyme activation, whereas dye 91 was mutagenic only in TA98 with S9 enzyme activation. These results indicated that dye 90 was a direct-acting mutagen. The results also suggested that removing one N-hydroxyethyl group and capping both –OH groups in the parent dye 80 did not affect mutagenicity, whereas removing both N-hydroxyethyl groups produced a strong direct-acting mutagen (dye 90) in all three bacterial strains. Increasing the length of the N-alkyl chain from two to three carbon atoms (dye 91) removed mutagenicity in TA98 without S9 activation. The results from TLC and HPLC analysis of product mixtures from rat liver and hamster liver enzyme S9 treatments of dye 80 and 89 confirmed that the metabolism of dye 80 involved de-hydroxyethylation of the substituted amino group. The analysis of product mixtures from rat liver S9 treatments of dye 92 indicated that the metabolism of dye 92 involved de-acetylation of the O-acetyl groups. Computational studies conducted in this research indicated that the shapes of HOMO and HOMO-1 and their energy differences did not provide a direct correlation between electronic properties and mutagenicity. Similarly, there was no correlation between log P values and mutagenicity.