Pyrazolopyridine derivatives as potent ASK1 inhibitors: design, synthesis, and biological evaluation

Currently, there is a lack of effective therapies for NASH, and ASK1 is considered a key therapeutic target. In this study, using the clinical candidate drug GS-4997 as a reference structure, a series of pyrazolopyridine-based ASK1 inhibitors were designed and synthesized through a fused-ring cyclization strategy and azacyclic modification of the benzamide aromatic ring. SAR studies identified three key pharmacophoric fragments: nitrogen substitution at the W-position of the core aromatic ring, an (S)-trifluoropropyl-modified triazole moiety, and a meta-isopropylpyrazole substitution on the left-hand aromatic ring. Based on these findings, compound 20 was constructed. This compound exhibited excellent ASK1 inhibitory activity (IC₅₀ = 6.3 nM), comparable to that of GS-4997 (IC₅₀ = 6.0 nM). In the concentration range of 0.5-5 μM, it showed no significant cytotoxicity toward LO2 cells, with cell viability consistently above 80 %, indicating a better safety profile than GS-4997. In a free fatty acid (FFA)-induced NASH cell model, compound 20 concentration-dependently alleviated lipid accumulation and significantly reduced T-CHO, TG, and LDL-C levels at concentrations of 1-6 μM (P < 0.05). Mechanistic studies revealed that compound 20 concentration-dependently inhibited TNF-α-induced activation of the ASK1-p38/JNK pathway, significantly reducing the p-ASK1/ASK1 ratio at a dose as low as 100 nM. Molecular docking results indicated that compound 20 forms key hydrogen bonds with VAL757 (1.90 Å) and LYS709 (2.27 Å) of ASK1, while its (S)-trifluoropropyl side chain further interacts with LYS688 (2.51 Å). These results demonstrate that compound 20 is a promising lead candidate for the treatment of NASH.