Non-alcoholic fatty liver disease (NAFLD) can be identified by the presence of macrovascular steatosis in the liver. The phenotypes of NAFLD range from nonalcoholic fatty liver to nonalcoholic steatohepatitis (NASH) [1]. NAFLD is becoming more common worldwide, and it is on course to become the primary cause of chronic liver disease in many parts of the world [2], [3]. It affects around one-fourth of the world's population and is fast becoming one of the primary causes of hepatocellular carcinoma (HCC), a common form of cancer and the largest cause of cancer-related deaths [4], [5]. The global increase in the prevalence of NAFLD patients necessitates the coordination of suitable healthcare measures. However, the lack of disruptive symptoms makes its earliest stages relatively undetected by people affected, allowing simple steatosis to advance to NASH and/or fibrosis, and eventually to HCC [6].

NASH-related HCC is a serious public health concern, and its prevalence is increasing in line with obesity, diabetes, and metabolic syndrome [7], [8]. Many variables promote the progression of NASH to NASH-related HCC (2 % of cases per year), including the tissue and immunological microenvironment, germline mutations in PNPLA3, and the microbiome [9]. NASH-HCC has distinct molecular and immunological characteristics when compared to other types of HCC and affects both men and women equally. Lipotoxicity, insulin resistance, and mitochondrial dysfunction all have role in NASH management, indicating a promising path for pharmacological study. However, the mechanism underlying the neoplastic transformation in NAFLD patients is unknown [10], [11], [12].

The enzyme pyruvate kinase (PKL) in liver cells is a major regulator of metabolic flux and ATP synthesis during glycolysis in liver cells. The PKLR gene encodes pyruvate kinase isoforms PKL and PKR in the liver and erythrocytes, respectively, and they are expressed at low levels in most other tissues where Pyruvate kinase M (PKM), takes their place [13], [14], [15]. It was suggested that PKLR could be an effective target for a NAFLD therapeutic strategy with low adverse effects on other human tissues. Previous research conducted by our group and an independent group established the significance of pyruvate kinase L/R (PKLR) in the development of NAFLD [15], [16]. Previous studies have demonstrated a positive correlation between PKLR overexpression/inhibition and key metabolic processes such as fatty acid synthesis, hepatic de novo lipogenesis (DNL), and triacylglyceride (TAG) levels in-vitro. Additionally, our findings introduce JNK-5A, identified through drug repositioning, which effectively reduces de novo lipogenesis and TAG levels by modulating PKL levels, without inducing side effects in both in-vivo and in-vitro experiments [14].

As a result, JNK-5A has been shown to work as a drug for the treatment of fatty liver disease. However, the molecular structure needs to be tuned to optimize the reduction of the expression levels of PKLR and other steatosis-related proteins as well as the reduction of triglyceride levels. To improve these findings, we aimed to design novel analogues of JNK-5A and evaluate their inhibitory effects on PKLR expression and lipid accumulation. Using computational techniques, novel JNK-3 inhibitors were designed, followed by the synthesis of a selected library of compounds. Then, comprehensive biological experiments were performed using several assays ranging from cell viability tests to the study of the modulation of the PKL pathway on the HepG2 cells, which were greatly impacted by the dual inhibitory activity of some compounds. Lastly, the biological activity of the promising compounds in preventing NAFLD was investigated.