Structure-activity relationship of steroidal-nitroxide hybrids: Dual-modulators of glucocorticoid receptor signalling and cellular redox state

Inflammation and redox imbalance are hallmarks of many diseases. Here, we report the synthesis and systematic evaluation of steroid-nitroxide hybrid compounds as dual-acting anti-inflammatory and redox-modulating agents. Hybrids were generated via ester (1a-c, 2a-b), amide (1d-h), and ether (3a-c) linkages at the C21 position of corticosteroids prednisolone, cortisol, and dexamethasone. The hybrids exhibit glucocorticoid receptor (GR) activity and suppress IL-6 secretion in vitro at variable potencies, some comparable to commercial corticosteroids. Cellular metabolic evaluation demonstrates various rates of intracellular cleavage for ester- and amide-linked hybrids, while ether-linked hybrids remain intact. Assessment of general reactive oxygen species (ROS) levels via the fluorogenic probe 2',7'-dichlorofluorescein diacetate (DCFDA) demonstrated linker-dependent pro-oxidative effects, with poorly labile and non-cleavable compounds (1a, 3a-c) causing sustained ROS elevation, while cleavable hybrids produced only transient, recoverable increases. Notably, one hybrid (1a) demonstrated higher ROS elevation in A549 cells over normal human fibroblasts (i.e. NFF cells) suggesting certain structural features of this hybrid induce specificity. Live-cell imaging using fluorescent analogues supports the mechanism of intracellular cleavage and efflux for cleavable hybrids, while non-cleavable hybrids remain within the cell. These findings demonstrate that steroidal-nitroxide hybrids harness both anti-inflammatory and redox modulation pathways, positioning them as a novel treatment strategy where dual anti-inflammatory and pro-oxidant strategies are advantageous.