Smaill J B, Fan J Y, Denny W A
Auckland Cancer Society Research Centre, Faculty of Medicine and Health Science, University of Auckland, New Zealand.
Anticancer Drug Des. 1998 Dec;13(8):857-80.
A series of bisbenzimidazoles bearing a variety of alkylating agents [ortho- and meta-mustards, imidazolebis(hydroxymethyl), imidazolebis(methylcarbamate) and pyrrolebis(hydroxymethyl)], appended by a propyl linker chain, were prepared and investigated for sequence-specificity of DNA alkylation and their cytotoxicity. Previous work has shown that, for para-aniline mustards, a propyl linker is optimal for cytotoxicity. Alkaline cleavage assays using a variety of different labelled oligonucleotides showed that the preferred sequences for adenine alkylation were 5'-TTTANANAANN and 5'-ATTANANAANN (underlined bases show the drug alkylation sites), with AT-rich sequences required on both the 5' and 3' sides of the alkylated adenine. The different aniline mustards showed little variation in alkylation pattern and similar efficiencies of DNA cross-link formation despite the changes in orientation and positioning of the mustard, suggesting that the propyl linker has some flexibility. The imidazole- and pyrrolebis(hydroxymethyl) alkylators showed no DNA strand cleavage following base treatment, indicating that no guanine or adenine N3 or N7 adducts were formed. Using the PCR-based polymerase stop assay, these alkylators showed PCR blocks at 5'-CG sites (the * nucleotide indicates the blocked site), particularly at 5'-TACGA 5'-AGCGGA, and 5'-AGCCGGT sequences, caused by guanine 2-NH2 lesions on the opposite strand. Only the (more reactive) imidazolebis(methylcarbamoyl) and pyrrolebis(hydroxymethyl) alkylators demonstrated interstrand cross-linking ability. All of the bifunctional mustards showed large (approximately 100-fold) increases in cytotoxicity over chlorambucil, with the corresponding monofunctional mustards being 20- to 60-fold less cytotoxic. These results suggest that in the mustards the propyl linker provides sufficient flexibility to achieve delivery of the alkylator to favoured (adenine N3) sites in the minor groove, regardless of its exact geometry with respect to the bisbenzimidazole carrier. The 'targeted' bisbenzimidazole bis(hydroxymethyl)pyrrole- and imidazole analogues showed very similar patterns of alkylation to the corresponding 'untargeted' compounds, with little evidence of additional selectivity imposed by this AT-preferring carrier.
制备了一系列带有各种烷基化剂（邻位和间位芥子气、咪唑双（羟甲基）、咪唑双（甲基氨基甲酸酯）和吡咯双（羟甲基））的双苯并咪唑，它们通过丙基连接链连接，并研究了其DNA烷基化的序列特异性及其细胞毒性。先前的研究表明，对于对苯胺芥子气，丙基连接链对细胞毒性是最佳的。使用各种不同标记的寡核苷酸进行的碱性切割试验表明，腺嘌呤烷基化的优选序列为5'-TTTANANAANN和5'-ATTANANAANN（下划线碱基显示药物烷基化位点），在烷基化腺嘌呤的5'和3'两侧都需要富含AT的序列。尽管芥子气的方向和位置发生了变化，但不同的苯胺芥子气在烷基化模式上几乎没有变化，并且在DNA交联形成方面具有相似的效率，这表明丙基连接链具有一定的灵活性。咪唑和吡咯双（羟甲基）烷基化剂在碱基处理后未显示DNA链断裂，表明未形成鸟嘌呤或腺嘌呤N3或N7加合物。使用基于PCR的聚合酶终止试验，这些烷基化剂在5'-CG位点（核苷酸表示阻断位点）显示PCR阻断，特别是在5'-TACGA、5'-AGCGGA和5'-AGCC*GGT序列处，这是由相反链上的鸟嘌呤2-NH2损伤引起的。只有（反应性更强的）咪唑双（甲基氨基甲酰）和吡咯双（羟甲基）烷基化剂表现出链间交联能力。所有双功能芥子气的细胞毒性比苯丁酸氮芥大幅增加（约100倍），相应的单功能芥子气的细胞毒性则低20至60倍。这些结果表明，在芥子气中，丙基连接链提供了足够的灵活性，以实现将烷基化剂递送至小沟中有利的（腺嘌呤N3）位点，而不管其相对于双苯并咪唑载体的确切几何形状如何。“靶向”双苯并咪唑双（羟甲基）吡咯和咪唑类似物显示出与相应的“非靶向”化合物非常相似的烷基化模式，几乎没有证据表明这种偏好AT的载体具有额外的选择性。
