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Biochemical Characterization of Some Pyrazolopyrimidine-Based Inhibitors of Xanthine Oxidase

Hemlata Tamta, Sukirti Kalra, and Anup K. Mukhopadhyay*

Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector 67, Phase X, S. A. S Nagar, Mohali, Punjab, India 160062; fax: 0091-172-214692; E-mail: akmukhopadhyay@niper.ac.in; Anup1niper@GMail.com

* To whom correspondence should be addressed.

Received April 25, 2005; Revision received June 25, 2005
Inhibition of xanthine oxidase-catalyzed conversion of xanthine to uric acid by various pyrazolopyrimidine-based inhibitors (allopurinol derivatives) was evaluated and compared with the standard inhibitor allopurinol. Three compounds out of the seven compounds used in the study were found to be reasonably good inhibitors of xanthine oxidase (XO). 4-Amino-6-mercaptopyrazolo-3,4-d-pyrimidine was found to be the most potent inhibitor of XO (IC50 = 0.600 ± 0.009 µM). 4-Mercapto-1H-pyrazolo-3,4-d-pyrimidine (IC50 = 1.326 ± 0.013 µM) and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine (IC50 = 1.564 ± 0.065 µM) also showed comparable inhibitory activity to that of allopurinol (IC50 = 0.776 ± 0.012 µM). All three compounds showed competitive type of inhibition with comparable Ki values. Induction of the electron transfer reaction catalyzed by XO in the presence of these compounds monitored as reduction of 2,6-dichlorophenolindophenol (DCPIP) revealed that electron transfer by 4-amino-6-mercaptopyrazolo-3,4-d-pyrimidine is comparable to that obtained by allopurinol or xanthine. However, 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine did not show DCPIP reduction. On the other hand, enzymatic reduction of cytochrome c in the presence of the three compounds was found to be insignificant and much less in comparison to allopurinol and xanthine. Therefore, both 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine and 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine displayed the inhibitory property and also did not produce XO-mediated reactive oxygen species (ROS). Since 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine was found to have some toxicity, the effect of 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine on the enzymatic formation of uric acid and ROS was investigated and it was found that this compound was inhibiting enzymatic generation of both uric acid and ROS. It can be noted that the standard inhibitor, allopurinol, inhibits uric acid formation but produces ROS.
KEY WORDS: two-electron reduction, one-electron reduction, competitive inhibition, IC50, 4-amino-6-mercaptopyrazolo-3,4-d-pyrimidine, 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine, 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine, allopurinol, xanthine, xanthine oxidase

DOI: 10.1134/S0006297906130086