Compounds and with potent ALR inhibitory

Compounds , and with potent ALR2 inhibitory activity were also tested for their inhibition ability against ALR1, as shown in . They all showed low activity with inhibition percentages no more than 45% at the concentration of 10μM, demonstrating good selectivity for ALR2. To evaluate the antioxidant properties of phenolic hydroxyl derivatives, the model reaction with the stable free radical of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was applied (). It was found that most of the compounds showed good DPPH radical scavenging activity ranging from 36.4 to 70.5% at the concentration of 10μM. Compounds were significantly active in the DPPH radical scavenging and more potent than the other series. Of tested compounds, with phenolic hydroxyl both at C7 position of the quinoxalinone core and C3 styryl ring showed the best scavenging activity, that is, 96.2%, 82.1%, and 70.5% at a concentration of 100μM, 50μM, and 10μM, respectively, which had an almost similar DPPH radical scavenging activity compared with the positive control of 6-hydroxy-2,5,7,8-chroman-2-carboxylic gpr109a inhibitor (Trolox) at higher concentration (). SAR studies of compounds , and revealed that the vinyl was more favored as the C3 spacer on the radical scavenging activity. Regarding to the effect of phenolic hydroxyl on the C3 side chain in compounds , the phenolic trihydroxyl compounds ( and ) were more active than the phenolic dihydroxyl compounds ( and ) and the single phenolic hydroxyl compounds ( and ). Moreover, the introduction of hydroxyl to the meta position of the C3 -hydroxystyryl ring as well as the C6 or C7 position of quinoxalinone core ( and ), or only methoxyl group to the C3 -hydroxystyryl meta position ( and ) of compounds and both largely boosted the DPPH radical scavenging activity. Therefore, the introduction of phenolic hydroxyl to quinoxalinone core and C3 side chain could successfully add antioxidant property to the ARI candidates. To get a better understanding of the mechanistic details and the above-described SARs, compound endowed with excellent activities both in the ALR2 inhibition and antioxidant reactions was docked with the conformation of the human ALR2-NADP-lidorestat complex (PDB code: 1Z3N). As shown in , docking results revealed that compound was tightly bound into the active site of ALR2. The carboxylate group was inserted deeply in the anion binding site by hydrogen-bonding interactions with the side chains of Tyr48 (3.02Å), His110 (2.78Å) and Trp111 (3.09Å) and by a stabilizing electrostatic interaction with the positively charged nicotinamide moiety of the NADP cofactor (NO=4.09Å). Besides, the oxygen atom of the C7-phenolic hydroxyl formed an additional hydrogen bond with the side chain of Trp20 (2.86Å), indicating that the introduction of phenolic hydroxyl to the core structure is beneficial for ALR2 inhibition. Moreover, the 3,4-dihydroxyphenyl ring of the C3 side chain was well paralleled to the indole ring of Trp 111 having a stable stacking interaction, and the 3-hydroxyl oxygen atom formed tight hydrogen bond with the side chain of Leu 300 (3.04Å), further confirming the importance of phenolic hydroxyl on C3 side chain. The C3-styryl ring was well placed into the specificity pocket lined by the side chains of Trp 111, Leu 300, Phe 122, Cys 303, Thr 113, Phe 115, and Trp79, while the quinoxalinone scaffold matched very well the hydrophobic pocket formed by the side chains of Leu300, Trp219, Phe122, Trp20 and Trp79. These interactions anchor the inhibitor tightly within the enzyme active site. The docking behavior of with the ALR1-NADP-fidarestat complex (PDB code: ) was also investigated. The result showed that the C3 styryl ring appeared to be mismatched, which was almost dissociated from the active site. Similar results were found from the docking of other compounds with relatively weaker activity against ALR1. These predictions are in accordance with the biological data in and well explain the selectivity for ALR2.