Optimized synthesis of 5N-D and the exploration of its role as a promising anti-aging agent via AKR1B1 inhibition and PI3K/Akt/Nrf2 regulation

An optimized synthesis for (R,E)-N-(3-(2-acetamido-3-(benzyloxy) propanamido) propyl)-2-cyano-3-(3,4-dihydroxyphenyl)acrylamide (5N-D) was reported here. 5N-D has been identified as a promising agent for treating age problems. Through in vitro enzyme testing, 5N-D is verified as an effective ALR2 inhibitor showing an IC₅₀ of 21.8 ± 1.5 nM, and excellent antioxidant showing an EC₅₀ of 2.8 ± 0.2 μM. Its selectivity index over ALR1 is 241.6. All these indexes are far better than the positive Epalrestat (EPA). 5N-D alleviates hyperglycemia-induced oxidative stress (OS) via AKR1B1 inhibition. It is identified not only as a non-enzymatic antioxidant, which interacts directly with Reactive Oxygen Species (ROS)/Reactive Nitrogen Species (RNS) and interrupts the free radical chain reactions; but also as an endogenous enzymatic antioxidant regulator, which regulates enzyme functions of superoxide catalase (CAT) and dismutase (SOD). 5N-D regulates PI3K/Akt/Nrf2 pathway to attenuate hyperglycemia-mediated mitochondrial superoxide overproduction in vitro. It ameliorates CuSO₄- and H₂O₂-induced OS in vivo. 5 N-D prolongs lifespan of C. elegans via the regulation of stress response genes such as PMK-1, DAF-16, AGE-1, AKT-1, AGE-1, SIR-2.1, and SKN-1. The results consistently support the potential of 5 N-D as an anti-aging agent.