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Aldose reductase (ALR2), one of the metabolically important enzymes, catalyzes the formation of sorbitol from glucose in the polyol pathway. ALR2 inhibition is required to prevent diabetic complications. In the present study, the novel bis-hydrazone compounds bearing isovanillin moiety (GY1-12) were synthesized, and various chromatographic methods were applied to purify the ALR2 enzyme. Afterward, the inhibitory effect of the synthesized compounds on the ALR2 was screened in vitro. All the novel bis-hydrazones demonstrated activity in na