Synthesis, pharmacological evaluation and mechanistic study of scutellarin methyl ester -4′-dipeptide conjugates for the treatment of hypoxic-ischemic encephalopathy (HIE) in rat pups.

• Scutellarin methyl ester-4′-dipeptide conjugates were synthesized and evaluated as potential protective agents against HIE. • 5k exhibit the highest P app A to B , lowest ER value in Caco-2 cells, and the highest uptake value in hPepT1-MDCK cells. • 5k exhibit more potent protective activity against oxidative damage by improving the relevant detection indicators. • 5k has significantly therapeutic effect on HIE rat pups by reducing infarct area and APP/ BACE-1 expression. • Favorable pharmacokinetic profiles of 5k were also confirmed in our studies. A series of novel scutellarin methyl ester-4′-dipeptide conjugates exhibiting active transport characteristics and protection against pathological damage caused by hypoxic-ischemic encephalopathy (HIE) were successfully designed and synthesized. The physiochemical properties of the obtained compounds, as well as the Caco-2 cell-based permeability and uptake into hPepT1-MDCK cells were evaluated using various analytical methods. Scutellarin methyl ester-4′-Val-homo-Leu dipeptide (5k) was determined as the optimal candidate with a high apparent permeability coefficient (P app A to B) of 1.95 ± 0.24 × 10−6 cm/s, low ER (P app BL to AP /P app AP to BL) of 0.52 in Caco-2 cells, and high uptake of 25.47 μmol/mg/min in hPepT1-MDCK cells. Comprehensive mechanistic studies demonstrated that pre-treatment of PC12 cells with 5k resulted in more potent anti-oxidative activity, which was manifested by a significant decrease in the malondialdehyde (MDA) and reactive oxygen species (ROS) levels, attenuation of the H 2 O 2 -induced apoptotic cell accumulation in the sub-G1 peak, and improvement in the expression of the relevant apoptotic proteins (Bcl-2, Bax, and cleave-caspase-3). Moreover, evaluation of in vivo neuroprotective characteristics in hypoxic-ischemic rat pups revealed that 5k significantly reduced infarction and alleviated the related pathomorphological damage. The compound was also shown to ameliorate the neurological deficit at 48 h as well as to decrease the brain tissue loss at 4 weeks. Conjugate 5k was demonstrated to reduce the amyloid precursor protein (APP) and β-site APP-converting enzyme-1 (BACE-1) expression. Pharmacokinetic characterization of 5k indicated favorable druggability and pharmacokinetic properties. The conducted docking studies revealed optimal binding of 5k to PepT1. Hydrogen bonding as well as cation-π interactions with the corresponding amino acid residues in the target active site were clearly observed. The obtained results suggest 5k as a potential candidate for anti-HIE therapy, which merits further investigation. [ABSTRACT FROM AUTHOR]