Intramolecular hydrogen bond interruption and scaffold hopping of TMC-5 led to 2-(4-alkoxy-3-cyanophenyl)pyrimidine-4/5-carboxylic acids and 6-(4-alkoxy-3-cyanophenyl)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-ones as potent pyrimidine-based xanthine oxidase inhibitors

Many pyrimidine-based Xanthine Oxidase (XO) inhibitors with diverse chemotypes have been reported recently. Our previous study revealed that 2-(4-alkoxy-3-cyano)phenyl-6-imino-1,6-dihydropyrimidine-5-carboxylic acid derivatives exhibited remarkable XO inhibitory potency. Notably, an intramolecular hydrogen bond (IMHB) formed between amino and carboxylic groups could be observed. With the hope to expand the structure-activity relationships (SARs) and obtain potential pyrimidine-based XO inhibitors, IMHB interruption and scaffold hopping were carried out on these compounds to design 2-(4-alkoxy-3-cyanophenyl)pyrimidine-4/5-carboxylic acids (11a-11n and 15a-15j) and 6-(4-alkoxy-3-cyanophenyl)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-ones (19a-19j). Among them, compound 19a (IC₅₀ = 0.039 μM) was identified as the most promising compound with substantially higher in vitro inhibitory potency than allopurinol (IC₅₀ = 7.590 μM) and comparable to febuxostat (IC₅₀ = 0.028 μM). The SAR analysis revealed that interrupting the IMHB through the removal of the amino group could damage the XO inhibitory potency; pyrimidine-4-carboxylic acid moiety was more beneficial for the XO inhibitory potency than the pyrimidine-5-carboxylic acid moiety. Additionally, Enzyme kinetics studies suggested that compounds 11a, 15a and 19a acted as mixed-type inhibitors for XO and the removal of 6-position amino group resulted in a weakened affinity to the free Enzyme, but an enhanced binding to the enzyme-substrate complex. Molecular modeling provided a reasonable explanation for the SARs observed in this study. Furthermore, in vivo hypouricemic effects demonstrated that compounds 15a and 19a could effectively reduce serum uric acid levels at an oral dose of 10 mg/kg, with 19a demonstrating a stronger effect than 15a. Therefore, our study proved that 6-(4-alkoxy-3-cyanophenyl)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-ones were potent pyrimidine-based XO inhibitors and compound 19a required further structural optimization as a potential and efficacious agents for the treatment of hyperuricemia and gout.