Drug Metabolism and Pharmacokinetics (S.W., B.C., D.Su., C.Z., S.B., Y.M., J.W., D.Sa., C.K., D.Z.), Discovery Chemistry (P.D., T.P., L.S., B.W.), and Discovery Biology (J.G., G.L.P.), Genentech, Inc., South San Francisco, California
Drug Metabolism and Pharmacokinetics (S.W., B.C., D.Su., C.Z., S.B., Y.M., J.W., D.Sa., C.K., D.Z.), Discovery Chemistry (P.D., T.P., L.S., B.W.), and Discovery Biology (J.G., G.L.P.), Genentech, Inc., South San Francisco, California.
Drug Metab Dispos. 2019 May;47(5):547-555. doi: 10.1124/dmd.118.085209. Epub 2019 Mar 11.
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 crosslinking, but it is difficult to analyze them for structure-activity correlation because of their DNA reactivity. To study their 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 analysis of DNA alkylation and shown 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 with the heat depurination method. 2) Di-adenine adducts were captured as expected for the CBI dimers, although the major type of adduct 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 nonalkylating 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.
双环丙基嘧啶并吲哚（包括环丙基吡咯并吲哚（CPI）或环丙基苯并吲哚（CBI））是一类 DNA 小沟烷化剂，而 sec o-CPI/CBIs 是合成前体，可以螺环化形成 CPI/CBI。双 CPI/CBIs 是潜在的药物候选物，因为它们通过 DNA 交联具有增强的细胞毒性，但由于其 DNA 反应性，很难对其进行结构-活性相关性分析。为了研究它们的 DNA 烷化作用，经常应用中性热水解来进行脱嘌呤处理。然而，据报道，在这种条件下会发生不需要的副反应，这可能导致 DNA 烷化数据与疗效结果之间相关性较差，尤其是对于双 CPI/CBIs。在这项研究中，开发了一种酸性脱嘌呤方法并应用于 DNA 烷化分析，结果表明与传统的中性热水解相比，该方法更简单、更温和。将三种双 sec o-CBI 与两种单 sec o-CPI 进行比较，对其 DNA 烷化和稳定性进行了表征。结果表明：1）与热脱嘌呤法相比，酸性脱嘌呤法能够捕获更多代表性的 DNA 加合物群体，有时甚至是不同的群体。2）二腺嘌呤加合物的捕获符合 CBI 二聚体的预期，尽管主要的加合物类型仍然是单腺嘌呤加合物。3）DNA 烷化率、DNA 加合物谱以及二加合物与单加合物的相对量受分子中非烷化部分的大小（可能还有亲脂性）的显著影响。4）螺环化和酰胺水解代表两种主要的降解途径。总的来说，通过应用酸性脱嘌呤分析，本研究阐明了新型双 sec o-CBI 的 DNA 加合物特征，主要为单烷化，并提供了一种评估 DNA 小沟烷化剂的替代方法。这些发现为评估 DNA 烷化剂和研究这些化合物的 DNA 烷化作用（一种处置机制）提供了有效的分析工具。
