Design, synthesis, and biological investigation of a new class of duocarmycin payloads for ADC development

Introduction: The duocarmycins belong to a class of agent that has been in the interest of chemist and drug developers for over four decades. Their pico-molar cellular potency, unique mechanism of action, and efficacy against drug-resistant cancer cells makes them attractive as payloads for inclusion in antibody-drug conjugate (ADC) discovery approaches. However, despite great advances in fine-tuning biological activity through structure-activity relationship (SAR) studies, no duocarmycin-based therapeutic has reached clinical approval. Recent duocarmycin-based ADCs under clinical evaluation have been hampered by a narrow therapeutic index, which have inspired us to modulate the duocarmycin scaffold designed by nature. Here we present our design approach, synthesis and biological investigation of new duocarmycin chemotypes. Methodology: Seco-duocarmycins derived from common scaffolds such as CBI and CPI are precursor molecules where the phenolic OH group essential for spirocyclisation was converted into a triflate. The latter was subjected to (i) Suzuki chemistry to generate novel aryl C-C linked compounds or (ii) or Buchwald-Hartwig reaction to generate aniline-linked duocarmycin target molecules. Subsequently, modulated compounds were studied for their ability to spirocyclise (LCMS), cause DNA damage using gamma-H2AX as marker and produce antiproliferative effects in a panel of breast, colon, ovarian, prostate cancer and rhabdomyosarcoma cell lines (MTT assay). Results: Biochemical assays revealed that these analogues exhibit a slower spirocyclization rate (2-48 hours) compared to the control compounds CPI-MI and CBI-MI (< 90 min). The library of duocarmycin payloads exhibited a wide range of cellular potencies in our panel of cancer cell lines (0.1 - 1000 nM), retaining antiproliferative activity in doxorubicin (MCF7adr) and docetaxel-resistant (PC3-D8) cancer cell lines whilst also be unaffected by the p53 status (HCT116 p53+/+ and p53-/-). Conclusion: New duocarmycin chemotype design offers an opportunity to reduce spirocyclisation rate and DNA reactivity to fine-tune cellular potency. We hypothesise incorporation of these promising payloads into ADC therapeutics will improve tumour biodistribution, bystander effect and reduce normal tissue toxicity.