Non-alcoholic steatohepatitis (NASH) is a common liver disease worldwide, with an estimated prevalence between 1.5 % and 6.5 % [1], which is a progressive form of nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, hepatocyte injury, and inflammation [1]. Especially in recent decades, the prevalence of liver cirrhosis resulting from NASH has sharply increased and will soon overtake viral hepatitis as the main indication for liver transplantation [2], [3], [4]. However, so far, no drug therapy has been approved for the treatment of NASH. Therefore, there is an urgent need to develop drugs to treat NASH at this stage.

Apoptosis signal regulated kinase 1 (ASK1, also known as MAP3K5) is a member of the mitogen activated protein kinase kinase kinases (MAP3Ks) family, located upstream of Jun N-terminal kinase (JNK) and p38 MAPK, and mediates their signaling pathway [5], [6]. After being activated by oxidative stress, ASK1 signals through the mitogen activated protein kinase pathway and terminates at the effector kinase p38 and c-Jun N-terminal kinase, which mediate pro-inflammatory and fibrotic changes in the liver [5], [7], [8], [9], [10]. The ASK1 pathway, as shown by liver expression of phosphorylated p-38 (p-p38), is upregulated in NASH patients and associated with liver fibrosis staging [10]. In preclinical models of NASH and liver fibrosis, ASK1 inhibition has been shown to have anti-fibrotic effects [9]. Therefore, ASK1 is a promising target for the treatment of NASH, and the development of new ASK1 inhibitors is of great significance to alleviate or even cure NASH.

The indole structure is an important multifunctional scaffold, and compounds containing indole structures have a wide range of biological activities, including anti-inflammatory [11], antibacterial [12], anticancer [13], anti-alzheimer [14], and analgesics [15]. Recently, Hu et al. [16] reported a series of novel ASK1 inhibitors with 1H-indazole moiety, and compound 1 was identified as a potent inhibitor with an IC50 value of 29.9 nM against ASK1 for the treatment of inflammatory bowel disease. Very recently, we have investigated and synthesized a novel series of compound structures in the early stage. These structures encompass hydrogenated indole, indole, benzimidazole, tetrahydroquinoxaline, benzoxazine, tetrahydroquinoline, phenol, nitrobenzene, pyridine, and quinolone moieties, designed as ASK1 inhibitors [17], [18]. GS-4997, developed by Gilead, is the only ASK1 inhibitor to enter clinical trials and is currently in Phase III clinical trials for the treatment of NASH [19]. GS-4997 has become a representative compound of ASK1 inhibitors and an important lead compound for structural modification of ASK1 inhibitors. Therefore, driven by our research focus, we endeavored to draw inspiration from the structural properties of compound 2 (GS-4997) and attempted to create an innovative indazole derivatives designed as an ASK1 inhibitor using a fused five-membered heterocycle and benzene ring, as shown in Fig. 1. Our concentration was directed towards examining the impact of R1, R2, R3, and the substitution of pyridine fragments on both activity and cytotoxicity. This investigation incorporated various analyses such as oil red O staining, biochemical assessments, exploration of the ASK1-p38/JNK signaling pathway mechanism, and molecular docking studies.