Combining two pharmacophores in a molecule can lead to useful synergistic effects. Herein, we show hybrid systems that combine sterically hindered phenols with dinitrobenzofuroxan fragments exhibit a broad range of biological activities. The modular assembly of such phenol/benzofuroxan hybrids allows variations in the phenol/benzofuroxan ratio. Interestingly, the antimicrobial activity only appears when at least two benzofuroxan moieties are introduced per phenol. The most potent of the synthesized compounds exhibit high cytotoxicity against human duodenal adenocarcinoma (HuTu 80), human breast adenocarcinoma (MCF-7), and human cervical carcinoma cell lines. This toxicity is associated with the induction of apoptosis via the internal mitochondrial pathway and an increase in ROS production. Encouragingly, the index of selectivity relative to healthy tissues exceeds that for the reference drugs Doxorubicin and Sorafenib. The biostability of the leading compounds in whole mice blood is sufficiently high for their future quantification in biological matrices.

The utility of sterically hindered phenols (SHPs) in drug design is based on their chameleonic ability to switch from an antioxidant that can protect healthy tissues to highly cytotoxic species that can target tumor cells. This work explores the biological activity of a family of 45 new hybrid molecules that combine SHPs equipped with an activating phosphonate moiety at the benzylic position with additional urea/thiourea fragments. The target compounds were synthesized by reaction of iso(thio)cyanates with C-arylphosphorylated phenols containing pendant 2,6-diaminopyridine and 1,3-diaminobenzene moieties. The SHP/urea hybrids display cytotoxic activity against a number of tumor lines. Mechanistic studies confirm the paradoxical nature of these substances which combine pronounced antioxidant properties in radical trapping assays with increased reactive oxygen species generation in tumor cells. Moreover, the most cytotoxic compounds inhibited the process of glycolysis in SH-SY5Y cells and caused pronounced dissipation of the mitochondrial membrane of isolated rat liver mitochondria. Molecular docking of the most active compounds identified the activator allosteric center of pyruvate kinase M2 as one of the possible targets. For the most promising compounds, 11b and 17b, this combination of properties results in the ability to induce apoptosis in HuTu 80 cells along the intrinsic mitochondrial pathway. Cyclic voltammetry studies reveal complex redox behavior which can be simplified by addition of a large excess of acid that can protect some of the oxidizable groups by protonations. Interestingly, the re-reduction behavior of the oxidized species shows considerable variations, indicating different degrees of reversibility. Such reversibility (or quasi-reversibility) suggests that the shift of the phenol-quinone equilibrium toward the original phenol at the lower pH may be associated with lower cytotoxicity.

This study describes synthesis and biological evaluation of a new phosphonamidate and a series of α-aminophosphonates, employing propargylamine as a key NH-nucleophilic synthon. Two synthetic approaches were successfully implemented: (i) the reaction with O-ethyl (3,5-di-tert-butyl-4-hydroxybenzyl)phosphonоchloridate to form a P–N bond; (ii) a 1,6-aza-Michael addition to dialkyl/diphenyl [(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dien-1-ylidene)methyl]phosphonate, affording the α-aminophosphonate scaffold via C–N bond formation. Initial in vitro cytotoxicity screening against a panel of human cancer cell lines using the MTT assay revealed that the phosphonamidate derivative exhibited the highest activity within the synthesized series.

A method was developed for the preparation of polychloroalkyl derivatives of diarylmethylphosphonates containing a sterically hindered phenol using 3-chloro-2,2-bis(chloromethyl)propanoyl chloride. Primary biological screening revealed low background cytotoxicity for most compounds, consistent with the profile of redox-activated prodrugs, while two derivatives exhibited moderate activity. The developed approach provides a basis for the targeted search for new selective anticancer agents based on modified sterically hindered phenols.

The convergence of ligand-based and structure-based computational strategies provides a robust foundation for hit optimization in anticancer drug discovery. We integrated ligand-based SAR with structure-based covalent docking to guide the optimization of haloacetamide-derived di(hetero)arylmethanes. SAR revealed that the 2-haloacetamide warhead is essential, while the di(hetero)aryl scaffold modulates activity, leading to submicromolar bis(pyrazolyl)methane hits. Rather than seeking to establish a definitive molecular target, we used GAPDH and GSTO1 as experimentally motivated target hypotheses to derive structural constraints for covalent scaffold design. Covalent docking was used to prioritize these hypotheses and guide the design of phosphorylated diarylmethane derivatives, leading to the identification of highly cytotoxic bromoacetamide compound, comparable to doxorubicin. In PANC-1 cells, this lead decreased LDH activity and increased PDH activity, suggesting a shift in cellular metabolism from glycolysis toward oxidative phosphorylation. The results demonstrate the utility of target-hypothesis-guided design under conditions in which molecular target identity remains unresolved.