Synthesis, SAR and Biophysical Mechanism of Action of Cyclopropabenzindole-Pyridinobenzodiazepine (CBI-PDD) Guanine-Adenine DNA Cross-Linking Agents

This study investigates the Structure–Activity Relationship (SAR) and biophysical mechanism of action of a series of novel Cyclopropabenzindole-Pyridinobenzodiazepine (CBI-PDD) agents designed to alkylate adenine and guanine bases in the DNA minor groove. A library of 18 analogs was synthesized using a modular approach to vary linker length, rigidity and the CBI and PDD core structures. Structural modifications included alkyl, aromatic, heteroaromatic and sterically constrained linkers, as well as prodrug variants. Several compounds demonstrated potent in vitro cytotoxicity, with lead analogs achieving IC₅₀ values in the low picomolar range across multiple human tumor cell lines. Control compounds with inactivated electrophilic groups in either or both pharmacophores showed markedly reduced activity, confirming the necessity of dual alkylation for optimal cytotoxic potency. Fluorescence-based thermal denaturation (ΔT_m up to 34 °C) and gel-based assays were used to assess DNA binding and provide mechanistic insights into the DNA sequence requirements for mono-alkylation and interstrand cross-linking. The study also includes a scalable synthesis of a linker-payload construct suitable for Antibody–Drug Conjugate (ADC) applications based on these agents. Overall, these findings support the use of CBI-PDDs as a promising class of sequence-selective DNA cross-linking agents for targeted cancer therapies.