Synthesis of New Shogaol Analogues as NRF2 Activators and Evaluation of Their Anti-Inflammatory Activity, Modes of Action and Metabolic Stability

Abstract

6-shogaol is a natural and the most potent bioactive vanilloid in dried Zingiber officinale rhizomes. Many scientific studies have reported the diverse biological activities of 6-shogaol. However, the major drawback of 6-shogaol is its instability at room temperature. We synthesised new shogaol thiophene compounds (STCs) by replacing the pentyl group in the sidechain with thiophene derivatives. The STCs were tested for their nuclear factor erythroid 2-related factor 2 (NRF2) activation ability in murine hepatoma cells (Hepa1c1c-7) by determining their NAD(P)H quinone oxidoreductase 1 (NQO1) inducing ability and expression of NRF2-associated antioxidant genes. The anti-inflammatory activity of STCs was determined in Escherichia coli lipopolysaccharide (LPSEc)-stimulated NR2-proficient and -silenced mouse microglial cells (BV-2) by measuring the inflammatory markers, cytokines, and mediators. The modes of action (interacting with the Kelch domain of KEAP1, covalent bonding with cysteines of KEAP1, and inhibition of GSK-3β enzyme activity) of NRF2 activation by STCs were determined using commercially available kits. The in vitro metabolic stability of the STCs in liver microsomes (humans, rats, and mice) was also investigated. The molecular docking and molecular dynamics studies were conducted to identify the binding poses, stability, and molecular interactions of the STCs in the binding pockets of Kelch and BTB domains of KEAP1 and GSK-3β enzyme. The new STCs were synthesised in good yields of > 85%, with a purity of about 95%, using a novel synthesis method by employing a reusable proline–proline dipeptide catalyst. The STCs are more potent than 6-shogaol in activating NRF2 and reducing inflammation. The nature of substituents on thiophene has a profound influence on the bioactivity of the STCs. Phenylthiophene STC (STC5) is the most potent, while thiophenes containing electron-withdrawing groups showed weaker bioactivity. The bioactivity of 6-shogaol is in the micromolar range, whereas STC5 showed bioactivity in the sub micromolar range. The STCs showed anti-inflammatory effects via NRF2-dependent and NRF2-independent mechanisms. The STCs improved NRF2 activity through multiple (KEAP1-independent and -dependent) mechanisms. The STCs showed decreased reactivity with thiols than 6-shogaol and thus may possess fewer side-effects than 6-shogaol. The STCs were more metabolically stable than 6-shogaol.