%0 Journal Article %A Shuai Wang %A Buyun Chen %A Peter Dragovich %A Thomas Pillow %A Leanna Staben %A Jun Guo %A Dian Su %A Chenghong Zhang %A Sudheer Bobba %A Yong Ma %A Jianshuang Wang %A Dewakar Sangaraju %A BinQing Wei %A Gail Lewis Phillips %A Cyrus Khojasteh %A Donglu Zhang %T A novel depurination methodology to assess DNA alkylation of chloro bis-seco-cyclopropylbenzoindoles (CBIs) allowed for comparison of minor groove reactivity %D 2019 %R 10.1124/dmd.118.085209 %J Drug Metabolism and Disposition %P dmd.118.085209 %X Duocarmycins (including cyclopropyl pyrroloindole, CPI or cyclopropyl benzoindole, CBI) are a class of DNA minor groove alkylators and seco-CPI/CBIs are synthetic pro-forms that can spirocyclize to CPI/CBI. Bis-CPI/CBIs are potential drug candidates because of their enhanced cytotoxicity from DNA cross-linking, but are difficult to be analyzed for structure-activity correlation because of their DNA reactivity. To study its DNA alkylation, neutral thermal hydrolysis has been frequently applied to process depurination. However, unwanted side reactions under this condition have been reported, which could lead to poor correlation of DNA alkylation data with efficacy results, especially for bis-CPI/CBIs. In this study, an acidic depurination method was developed and applied for analyses of DNA alkylation, and showed to be an easier and milder method than the traditional neutral thermal hydrolysis. DNA alkylation and stability of three bis-seco-CBIs were characterized in comparison with two mono-seco-CPIs. The results suggested that: (1) The acidic depurination method was capable of capturing a more representative population, sometimes a different population, of DNA adducts as they existed on DNA compared to the heat depurination method. (2) Di-adenine adducts were captured as expected for the CBI dimers, while the major type of adducts was still mono-adenine adducts. (3) The rate of DNA alkylation, DNA adduct profile, and relative amounts of di-adduct versus mono-adduct were significantly affected by the size, and possibly lipophilicity of the non-alkylating part of the molecules. (4) Spirocyclization and amide hydrolysis represented two major pathways of degradation. Overall, by applying acidic depurination analyses, this study has illustrated DNA adduct characteristics of novel bis-seco-CBIs with dominating mono-alkylation and provides an alternative method for evaluating DNA minor groove alkylators. These findings provide an effective analytical tool to evaluate DNA alkylators and to study the DNA alkylation that is a disposition mechanism of these compounds. %U https://dmd.aspetjournals.org/content/dmd/early/2019/03/11/dmd.118.085209.full.pdf