Necroptosis is a type of programmed cell death that was first proposed in 2005 and is associated with a variety of inflammatory diseases [1]. Necroptosis has received increasing attention as it is accompanied by cell swelling, rupture of the plasma membrane, and efflux of cellular contents, and leads to a variety of diseases, including systemic inflammation, neurodegenerative diseases, autoimmune diseases, and cancers [[2], [3], [4], [5], [6]]. Receptor Interacting Protein Kinase1 (RIPK1) is a crucial upstream kinase that plays a significant regulatory role in necroptosis, mediating the balance between cell survival and death [7]. Activation of RIPK1 has been demonstrated in a variety of diseases, while inhibition of RIPK1 activity has shown efficacy in a variety of disease models, including inflammatory diseases, acute ischemic injury, and neurodegenerative diseases [8,9]. TNF-α-induced necroptosis is the most commonly studied model, and RIPK1 is a major regulator of TNFR1-mediated activation of the pro-survival nuclear factor kappa-B (NF-kB), pro-apoptotic FADD-caspase-8, and pro-necrotic RIPK3-MLKL signaling pathways. Binding of TNF-α to its receptor TNFR1 leads to homotrimerization of TNF receptor 1 (TNFR1), which then recruits TNFR1-associated death domain protein (TRADD), RIPK1, TNF receptor-associated factor 2 (TRAF2), cellular inhibitors of apoptosis1/2 (cIAP1/2) to form complex I [2]. When RIPK1 is ubiquitinated it activates the NF-kB pathway to promote cell survival [10,11]. If cIAP1/2 activity is inhibited, it blocks RIPK1 ubiquitination and separates TRADD and RIPK1 from TNFR1. TRADD separates from TNFR1 and recruits FADD and pro-caspase-8 to form complex IIa, which promotes apoptosis. Phosphorylation of RIPK1/RIPK3 recruits and promotes MLKL phosphorylation, leading to necroptosis [12,13], which triggers the release of cellular contents and associated molecular patterns (DAMP), leading to necroptosis and thus inflammation. Therefore, targeting the necroptotic pathway caused by RIPK1 is important for the treatment of inflammatory diseases (see Fig. 1).

Nowadays, numerous RIPK1 inhibitors have been reported, yet only a limited number have proceeded to clinical trials. Nec-1 was the first necroptosis inhibitor [1], which was of great significance for subsequent research and development, but its micromolar low potency and poor metabolic stability limited its further investigation. GlaxoSmithKline developed GSK2982772 and has good activity and kinase selectivity, and it has been used in clinical studies for a variety of chronic inflammatory diseases, including psoriasis, ulcerative colitis, and rheumatoid arthritis [1,14,15]. Structural modification based on GSK2982772 yielded GSK3145095, which also has excellent activity and was used in phase I clinical studies in pancreatic cancer and some advanced solid tumors, but clinical studies have been discontinued [16]. There are also some other reported RIPK1 inhibitors (Fig. 2), including Nec-1 [1], Nec-1s [17], PK68 [18], Cpd 72 [19], GSK963 [20], MBM105 [21].

It is reported that GSK963 has certain inhibitory activity against RIPK1. To develop a more effective RIPK1 inhibitor, we made strategic modifications to the parent nucleus structure by introducing an oxygen atom adjacent to the nitrogen atom, thereby creating an isoxazoline framework. Among the identified inhibitors, compound 22 demonstrated significant anti-necroptotic activity (EC50 = 30.0 nM) and exhibited potent enzymatic inhibition against RIPK1 (EC50 = 6.9 nM). Furthermore, it effectively mitigated TNF-α-induced inflammation and organ damage.