Spinal cord injury (SCI) is a serious traumatic neurological injury disease that occurs primarily as a result of car accidents and falls [1]. It has a high rate of disability and mortality, causing long-term psychological stress and financial burden to both patients and their families [2]. At present, in clinical practice, the treatment of spinal cord injury mainly includes surgical decompression, medication, hyperbaric oxygen therapy [3] and prosthetic rehabilitation, etc. However, due to its very complex pathophysiological process and microenvironmental imbalance, the current treatment effect is not satisfactory [4]. The pathophysiologic processes are categorized into primary and secondary injury, with primary injury being irreversible [5]. Secondary injury is manifested by post-traumatic inflammatory response, free radical formation, programmed cell death, edema and glutamate excitotoxicity [6], this change is reversible. Therefore controlling secondary injury after SCI may be the key to treating spinal cord injuries as well as a treatment for spinal cord injuries.

Programmed cell death (PCD) includes autophagy, necrotic apoptosis, pyroptosis, and ferroptosis [7]. Ferroptosis is a novel mode of cell death that is dependent on iron and reactive oxygen species (ROS) and is caused by an imbalance in the production and degradation of intracellular lipid reactive oxygen species [8]. And glutamate can also induce ferroptosis [9]. When intracellular iron is overloaded, Fe2+ undergoes a Fenton reaction with hydrogen peroxide (H2O2), generating hydroxyl radicals that increase intracellular ROS levels and promote lipid peroxidation, thereby causing ferroptosis [10]. PUFA undergoes acylation and lipidation, followed by non-enzymatic and enzymatic reactions to generate ROS, Malondialdehyde (MDA) and 4-Hydroxynonenal (4-HNE) leading to severe cytotoxicity and thus ferroptosis [11]. Furthermore, GPX4, a selenocysteine-containing and GSH-dependent enzyme, is a key regulator of ferroptosis. It converts GSH to Glutathione Oxidized (GSSG) and reduces lipid peroxidation, thereby reducing the formation of ROS and lipid peroxides and oxidative stress damage, and ultimately inhibiting ferroptosis [12].

In recent years, a large body of literature has shown that ferroptosis is associated with many neurodegenerative diseases, such as ischemic stroke [13], hemorrhagic stroke [14], Alzheimer's disease [15], Parkinson's disease [16], traumatic brain injury, and spinal cord injury [17]. It has been shown that ROS production and lipid peroxidation are important causes of secondary damage after SCI [18], and both iron overload [19] and glutamate accumulation [20] associated with ferroptosis are also present in SCI, so ferroptosis inhibitors are expected to improve SCI.

A variety of synthetic ferroptosis inhibitors have been tested in animal models, such as Ferrostatin-1 in rat cerebral ischemia–reperfusion [21] and thioacetamide-induced acute liver injury models [22], Liproxstatin-1 in mice with ischemia/reperfusion-induced acute renal injury [23], Deferoxamine in a mouse model of osteoarthritis [24], UAMC-3203 in a rat model of cardiac arrest [25], and SRS11-92 in a rat model of cerebral ischemia/reperfusion [26]. However, there is no inhibitor of ferroptosis that has been applied to clinical use so far, so there is an urgent need to discover a better inhibitor of ferroptosis.

In previous research, Yang's team has developed a series of novel ferroptosis inhibitors with phenothiazine scaffold. Compounds 1, 12a, and 51, specifically, have demonstrated excellent ferroptosis inhibition activity, attributed to the presence of piperazine ring and analogs in these compounds (Fig. 1A) [27]. However, these compounds exhibited significant hERG block [28]. Current literature suggests effective methods to diminish hERG inhibitory effects of drugs include reducing lipophilicity (clogP) [29] and decreasing basicity (pKa) [30], [31]. Yang et al. found that introducing the vinyl group at the 2-position of 10H-phenothiazine (compound 2 and 7 m) reduced the hERG inhibition activity, probably due to the reduced lipophilicity of the compound (Fig. 1B) [28]. Furthermore, substituting the amino group with an amide or sulfonamide can lower the basicity of the compound, which is advantageous in reducing hERG inhibitory activity (Fig. 1B) [30], [31]. Based on these investigations, we would like to design and synthesize a type of phenothiazine derivatives with higher ferroptosis inhibitory activity and lower hERG inhibition activity. Therefore, we expect to introduce vinyl groups in region I and sulfonyl groups in region II to reduce hERG inhibition activity, while piperazine and its analogs are introduced in region III to enhance ferroptosis inhibitory activity (Fig. 1C). Based on the above ideas, 24 novel sulfonamides inhibitors of ferroptosis were designed and synthesized. As a result, compound 23b was found to have the best activity in erastin-induced PC12 cells (EC50 = 0.001 μM) and was found to have low hERG inhibition activity (IC50 > 30 μM). Furthermore, we elucidated the mechanism of action of compound 23b and evaluated the therapeutic effect of compound 23b in an SD rat model of SCI, and determined the in vivo and in vitro toxicity of compound 23b. Finally we also performed a pharmacokinetic evaluation of compound 23b.