In our effort to develop potent anti-hyperglycemic compounds with inhibitory activity against α-amylase and α-glucosidase, a series of novel quinoxaline-isoxazole moieties were synthesized. The novel quinoxaline-isoxazole derivatives were assessed in vitro for their anti-hyperglycemic activities on α-amylase and α-glucosidase inhibitions. The results revealed promising IC50 values compared to acarbose as a positive control for α-amylase and α-glucosidase. Among them, N-Ethyl-7-chloro-3-((3-phenylisoxazol-5-yl)methoxy)quinoxalin-2-amine 5b showed dual inhibitory with IC50 of 24.0 µM for α-amylase and 41.7 µM for α-glucosidase. In addition, N-Ethyl-7-methoxy-3-((3-(2-chlorophenyl)isoxazol-5-yl)methoxy)quinoxalin-2-amine 5j also had dual bioactivities against α-amylase and α-glucosidase with IC50 of 17.0 and 40.1 µM, respectively. Nevertheless, two more compounds N-Ethyl-7-cyano-3-((3-phenylisoxazol-5-yl)methoxy)quinoxaline-2-amine 5e showed strong mono-inhibition for α-glucosidase with IC50 of 16.6 µM followed by N-Ethyl-7-methoxy-3-((3-phenylisoxazol-5-yl)methoxy)quinoxalin-2-amine 5 f with IC50 of 18.6 µM. The molecular docking study for α-glucosidase inhibitor provided the binding energy ranging from 8.3 to 9.1 kcal/mol and α-amylase inhibitor showed the binding energy score at 8.4 and 8.5 kcal/mol. The dual inhibitions nature of 5b and 5j were further analyzed and confirmed via molecular dynamics including the stability of the compound, interaction energy, binding free energy, and the interaction residue analysis using the MM-GBSA approach. The results showed that compound 5j was the most potent compound. Lastly, the drug-likeness properties were also evaluated with all synthesized compounds 5a-5j and the results reveal that all potent compounds meet Lipinski's rules of five.
New phenylisoxazole quinoxalin-2-amine hybrids 5a-i were successfully synthesised with yields of 53-85% and characterised with various spectroscopy methods. The synthesised hybrids underwent in vitro α-amylase and α-glucosidase inhibitory assays, with acarbose as the positive control. Through the biological study, compound 5h exhibits the highest α-amylase inhibitory activity with IC50 = 16.4 ± 0.1 μM while compounds 5a-c, 5e and 5h exhibit great potential as α-glucosidase inhibitors, with 5c being the most potent (IC50 = 15.2 ± 0.3 μM). Among the compounds, 5h exhibits potential as a dual inhibitor for both α-amylase (IC50 = 16.4 ± 0.1 μM) and α-glucosidase (IC50 = 31.6 ± 0.4 μM) enzymes. Through the molecular docking studies, the inhibition potential of the selected compounds is supported. Compound 5h showed important interactions with α-amylase enzyme active sites and exhibited the highest binding energy of -8.9 ± 0.10 kcal mol-1, while compound 5c exhibited the highest binding energy of -9.0 ± 0.20 kcal mol-1 by forming important interactions with the α-glucosidase enzyme active sites. The molecular dynamics study showed that the selected compounds exhibited relative stability when binding with α-amylase and α-glucosidase enzymes. Additionally, compound 5h demonstrated a similar pattern of motion and mechanism of action as the commercially available miglitol.