3,4-Dihydroxy-5-nitrobenzaldehyde (DHNB) is a potent inhibitor of xanthine oxidase: A potential therapeutic agent for treatment of hyperuricemia and gout

Abstract

Hyperuricemia, excess of uric acid in the blood, is a clinical problem that causes gout and is also considered a risk factor for cardiovascular disease. The enzyme xanthine oxidase (XO) produces uric acid during the purine metabolism; therefore, discovering novel XO inhibitors is an important strategy to develop an effective therapy for hyperuricemia and gout. We found that 3,4-dihydroxy-5-nitrobenzaldehyde (DHNB), a derivative of the natural substance protocatechuic aldehyde, potently inhibited XO activity with an IC50 value of 3μM. DHNB inhibited XO activity in a time-dependent manner, which was similar to that of allopurinol, a clinical XO inhibitory drug. DHNB displayed potent mixed-type inhibition of the activity of XO, and showed an additive effect with allopurinol at the low concentration. Structure–activity relationship studies of DHNB indicated that the aldehyde moiety, the catechol moiety, and nitration at C-5 were required for XO inhibition. DHNB interacted with the molybdenum center of XO and was slowly converted to its carboxylic acid at a rate of 10−10mol/L/s. In addition, DHNB directly scavenged free radical DPPH and ROS, including ONOO− and HOCl. DHNB effectively reduced serum uric acid levels in allantoxanamide-induced hyperuricemic mice. Furthermore, mice orally given a large dose (500mg/kg) of DHNB did not show any side effects, while 42% of allopurinol (500mg/kg)-treated mice died and their offspring lost their fur. Thus, DHNB could be an outstanding candidate for a novel XO inhibitory drug that has potent activity and low toxicity, as well as antioxidant activity and a distinct chemical structure from allopurinol.