Salt form and phosphate modification of an SN-38-nitrogen mustard conjugate: Overcoming water solubility limitations,...

Cancer therapy has long faced a critical bottleneck wherein monotherapy often proves insufficient due to tumor heterogeneity and compensatory mechanisms, while combination chemotherapy frequently leads to localized drug ratio mismatch and exacerbated toxicity owing to differential in vivo drug distribution. To fundamentally address this challenge, we focused on achieving precise combined therapy at the single-molecule level. The core of this strategy involves designing novel bifunctional molecules that integrate the Topo I inhibitor SN-38 and the alkylating agent nitrogen mustard via a covalent linkage, with further prodrug modification using salt forms and phosphate ester groups to optimize their physicochemical properties. Among the candidate molecules, MN33-47 and MN33-63 demonstrated outstanding performance, not only significantly improving the solubility of SN-38 but also exhibiting broad-spectrum antiproliferative activity superior to individual drugs or their physical mixtures across various cancer cell models. Mechanistic studies further revealed that these molecules inhibit Topo I and promote its degradation, while simultaneously inducing DNA crosslinking, thereby combinedly enhancing DNA damage. This cascade of events subsequently leads to G₂/M phase cell cycle arrest and inhibition of cell migration. Subsequent downregulation of Bcl-2 protein relieves its suppression of the mitochondrial apoptosis pathway, ultimately activating caspase-3 and effectively initiating the apoptosis program. In CT26 tumor-bearing mouse models, MN33-47 and MN33-63 exhibited significant dose-dependent tumor suppression without causing apparent toxic side effects, demonstrating superior safety compared to combination chemotherapy. In summary, this work provides a practical solution to overcome the clinical limitations of SN-38 and nitrogen mustard, primarily through molecular innovation that "solidifies" combination therapy into a single entity, thereby directly resolving the inherent conflict between enhancing efficacy and exacerbating toxicity.